Humanized antibodies against monocyte chemotactic proteins

ABSTRACT

The invention provides humanized antibodies that bind to a plurality of β-chemokines, particularly monocyte chemotactic proteins MCP-1, MCP-2 and MCP-3. The invention also provides therapeutic reagents and methods of treating disorders associated with detrimental MCP activity.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.60/430,007, filed Nov. 27, 2002. This application is related to PCTapplication no. PCT/US02/38229 filed Nov. 27, 2002. This application isalso related to U.S. Provisional Application No. 60/343,391, filed Nov.30, 2001. This application is also related to U.S. ProvisionalApplication No. 60/383,277, filed May 24, 2002. This application is alsorelated to U.S. Provisional Application No. 60/400,469, filed Aug. 1,2002. The entire contents of each of these patents and patentapplications are hereby incorporated herein by reference.

BACKGROUND OF THE INVENTION

“Chemokines,” which take their name from chemotactic cytokines, aresmall secreted polypeptides that regulate movement of immune cells intotissues (Baggiolini et al. (1994) Adv. Immunol. 55:97-179; Oppenheim etal. (1991) Ann Rev. Immunol. 9:617-648). Chemokines are assigned tothree different families based on the number and position of conservedcysteine residues (Van Coillie et al. (1999) Cytokine & Growth FactorRev. 10:61-86). The a and b chemokines each contain four conservedcysteine residues. The first two cysteines of the a chemokines areseparated by a single amino acid, thus containing a CXC amino acidmotif. The first two conserved cysteines of the b chemokines areadjacent. Thus, the b chemokines are also known as C—C chemokines. Bycontrast, lymphotactin is the sole member of the third family ofchemokines, and contains only the second and fourth conserved cysteineresidues. Interestingly, in humans, a chemokines are all encoded bygenes on chromosome 4, b chemokines are all encoded by genes onchromosome 17, and lymphotaxin is encoded by genes on chromosome 1.

The b-chemokines form a gradient that serves as a chemoattractant andpotential proliferation signal for immune and other cells such asmonocytes, macrophages, basophils, eosinophils, T lymphocytes andfibroblasts. MCP-1, MCP-2 and MCP-3 share sequence homology with oneanother at the amino acid level. Through interaction with specificreceptors, termed C—C chemokine receptors (CCR) which are G-proteincoupled, seven transmembrane receptors (Rossi and Zlotnik (2000) Ann.Rev. Immunol. 18:217-242), the b-chemokines regulate the expression ofadhesion molecules on endothelial cells and thereby indirectly affectdiapedesis and extravasation of immune cells from the circulation intotissues. There are ten different CCRs (CCR1 through CCR10). CCR2 acts asa receptor for MCP-1, MCP-2, MCP-3, and MCP-4 (Rossi and Zlotnik (2000)Ann. Rev. Immunol. 18:217-242). However, all human MCPs have been shownto interact with more than one receptor (Van Coillie et al. (1999)Cytokine & Growth Factor Rev. 10:61-86).

Human MCP-1, MCP-2 and MCP-3 all have chemotactic activity for a varietyof cell types, including T lymphocytes and monocytes (Van Coillie et al.(1999) Cytokine & Growth Factor Rev. 10:61-86). Other shared functionsof MCP-1, MCP-2, and MCP-3 include induction of N-acetylb-D-glucosaminidase release, gelatinase B release, and granzyme Arelease which are believed to help the cells digest the extracellularmatrix components necessary to enable them to migrate into tissues (VanCoillie et al. (1999) Cytokine & Growth Factor Rev. 10:61-86). Inaddition, MCP-1 and MCP-3 share various functions, such as induction ofarachidonic acid release and stimulation of a respiratory burst (VanCoillie et al. (1999) Cytokine & Growth Factor Rev. 10:61-86).

MCP-1-specific antibodies have previously been described in theliterature (WO 01/89582, WO 01/89565, Luo et al. (1994) J Immunol153:3708-16; Traynor, et al (2002) J Immunol 168:4659-66). Certain MCP-1antibodies have been described as binding MCP-1 and MCP-3, specificallythe MRHAS domain of MCP-1 and MCP-3 (WO 95/09232). In addition, a humananti-MCP-1 antibody has also been described (WO 02/02640). There is aneed in the art to identify antibodies which can be used to manipulateb-chemokines in general, and to specifically modulate the activity ofmultiple chemokines, e.g., MCP-1 and MCP-2 or MCP-3.

SUMMARY OF THE INVENTION

The present invention features new immunological reagents, inparticular, therapeutic antibody reagents for the prevention andtreatment of disorders associated with detrimental MCP activity. Theinvention is based, at least in part, on the identification andcharacterization of two monoclonal pan-antibodies that specifically bindto MCPs and are effective at binding MCPs, including MCP-1 and MCP-2,with high affinity and at inhibiting MCP-induced chemotaxis. Structuraland functional analysis of these antibodies leads to the design ofvarious humanized antibodies for prophylactic and/or therapeutic use. Inparticular, the invention features humanization of the variable regionsof these antibodies and, accordingly provides for humanizedimmunoglobulin or antibody chains, intact humanized immunoglobulins orantibodies, and functional immunoglobulin or antibody fragments, inparticular, antigen binding fragments, of the featured antibodies.

Polypeptides comprising the complementarity determining regions of thefeatured monoclonal antibodies are also disclosed, as are polynucleotidereagents, vectors and host suitable for encoding said polypeptides.

Methods of treatment of disorders associated with detrimental MCPactivity are disclosed, as are pharmaceutical compositions and kits foruse in such applications.

Also featured are methods of identifying residues within the featuredmonoclonal antibodies which are important for proper immunologicfunction and for identifying residues which are amenable to substitutionin the design of humanized antibodies having improved binding affinitiesand/or reduced immunogenicity, when used as therapeutic reagents.

In one embodiment, the invention features a humanized immunoglobulinheavy chain or antigen-binding fragment thereof comprising variableregion complementary determining regions (CDRs) from the 11K2immunoglobulin heavy chain variable region sequence set forth as SEQ IDNO: 27, and variable framework regions from a human acceptorimmunoglobulin heavy chain sequence, provided that at least oneframework residue is substituted with the corresponding amino acidresidue from the mouse 11K2 heavy chain variable region sequence,wherein the framework residue is selected from the group consisting of:

a residue that non-covalently binds antigen directly;

a residue adjacent to a CDR;

a CDR-interacting residue; and

a residue participating in the VL-VH interface.

In another embodiment, the invention features a humanized immunoglobulinlight chain or antigen-binding fragment thereof comprising variableregion complementary determining regions (CDRs) from the 11K2immunoglobulin light chain variable region sequence set forth as SEQ IDNO: 28 and variable framework regions from a human acceptorimmunoglobulin light chain, provided that at least one framework residueis substituted with the corresponding amino acid residue from the mouse11K2 light chain variable region sequence, wherein the framework residueis selected from the group consisting of:

a residue that non-covalently binds antigen directly;

a residue adjacent to a CDR;

a CDR-interacting residue; and

a residue participating in the VL-VH interface.

In some embodiments, a CDR-interacting residue is identified by modelingthe 11K2 heavy chain based on the solved structure of a murineimmunoglobulin heavy chain that shares at least 70%, 80%, or 90%sequence identity with the 11K2 heavy chain. In other embodiments, aCDR-interacting residue is identified by modeling the 11K2 light chainbased on the solved structure of a murine immunoglobulin light chainthat shares at least 70%, 80%, or 90% sequence identity with the 11K2light chain.

Another embodiment of the invention features a humanized immunoglobulinheavy chain or antigen-binding fragment thereof comprising variableregion complementary determining regions (CDRs) from the 11K2immunoglobulin heavy chain variable region sequence set forth as SEQ IDNO: 27, and variable framework regions from a human acceptorimmunoglobulin heavy chain sequence, provided that at least oneframework residue is substituted with the corresponding amino acidresidue from the mouse 11K2 heavy chain variable region sequence,wherein the framework residue is a residue capable of affecting heavychain variable region conformation or function as identified by analysisof a three-dimensional model of the variable region.

In yet other embodiments, the invention describes a humanizedimmunoglobulin light chain or antigen-binding fragment thereofcomprising variable region complementary determining regions (CDRs) fromthe 11K2 immunoglobulin light chain variable region sequence set forthas SEQ ID NO: 28, and variable framework regions from a human acceptorimmunoglobulin light chain, provided that at least one framework residueis substituted with the corresponding amino acid residue from the mouse11K2 light chain variable region sequence, wherein the framework residueis a residue capable of affecting light chain variable regionconformation or function as identified by analysis of athree-dimensional model of the variable region.

In some embodiments, the framework residue is selected from the groupconsisting of a residue capable of interacting with antigen, a residueproximal to the antigen-binding site, a residue capable of interactingwith a CDR, a residue adjacent to a CDR, a residue within 6 Å of a CDRresidue, a canonical residue, a vernier zone residue, an interchainpacking residue, and a rare residue. In other embodiments, the frameworkresidue is selected from the group consisting of a residue capable ofinteracting with antigen, a residue proximal to the antigen-bindingsite, a residue capable of interacting with a CDR, a residue adjacent toa CDR, a residue within 6 Å of a CDR residue, a canonical residue, avernier zone residue, an interchain packing residue, and an unusualresidue.

In still other embodiments, the framework residue is identified bymodeling the 11K2 heavy chain based on the solved structure of a murineimmunoglobulin heavy chain that shares at least 70%, 80%, or 90%sequence identity with the 11K2 heavy chain. In still other embodiments,the framework residue is identified by modeling the 11K2 light chainbased on the solved structure of a murine immunoglobulin light chainthat shares at least 70%, 80%, or 90% sequence identity with the 11K2light chain.

In one embodiment, the invention features a humanized antibody orantigen-binding fragment thereof comprising the complementarydetermining regions (CDR1, CDR2 and CDR3) of the 11K2 variable heavychain sequence set forth as SEQ ID NO: 27. The invention also features ahumanized antibody comprising the complementary determining regions(CDR1, CDR2 and CDR3) of the 11K2 variable light chain sequence setforth as SEQ ID NO: 28. In still another embodiment, the inventionfeatures a humanized antibody, or antigen-binding fragment thereof,which specifically binds to MCP-1 comprising variable region comprisingcomplementary determining regions (CDRs) corresponding to CDRs from themouse 11K2 antibody. In some embodiments, the fragment of the inventionis a Fab fragment.

In yet another embodiment, the invention features a chimericimmunoglobulin comprising a variable region sequence substantially asset forth in SEQ ID NO: 27 or SEQ ID NO: 28, and constant regionsequences from a human immunoglobulin.

In one embodiment, the invention features a humanized antibodycomprising the complementary determining regions (CDR1, CDR2 and CDR3)of the 11K2 variable heavy chain sequence set forth as SEQ ID NO: 27. Inanother embodiment, the invention features a humanized antibodycomprising the complementary determining regions (CDR1, CDR2 and CDR3)of the 11K2 variable light chain sequence set forth as SEQ ID NO: 28.

In another embodiment, the invention features a humanized antibody, orantigen-binding fragment thereof, which specifically binds to MCP-1comprising variable region comprising complementary determining regions(CDRs) corresponding to CDRs from the mouse 11K2 antibody. The inventionalso describes a chimeric immunoglobulin comprising a variable regionsequence substantially as set forth in SEQ ID NO: 27 or SEQ ID NO: 28,and constant region sequences from a human immunoglobulin.

In yet another embodiment, the invention features a method foridentifying residues amenable to substitution in a humanized 11K2immunoglobulin variable framework region, comprising modeling thethree-dimensional structure of the 11K2 variable region based on asolved immunoglobulin structure and analyzing said model for residuescapable of affecting 11K2 immunoglobulin variable region conformation orfunction, such that residues amenable to substitution are identified.The invention also features use of the variable region sequence setforth as SEQ ID NO: 27 or SEQ ID NO: 28, or any portion thereof, inproducing a three-dimensional image of a 11K2 immunoglobulin, 11K2immunoglobulin chain, or domain thereof.

In still another embodiment, the invention features a method of treatinga disorder associated with detrimental MCP activity in a subject byadministering a nucleic acid molecule that encodes an immunoglobulinheavy chain comprising the amino acid sequence of SEQ ID NO: 47 or theamino acid sequence of SEQ ID NO: 48 and a nucleic acid molecule thatencodes an immunoglobulin light chain comprising the amino acid sequenceof SEQ ID NO: 49 or the amino acid sequence of SEQ ID NO: 50, underconditions such that said immunoglobulin chains are expressed, therebytreating the subject.

In one embodiment, the invention features a humanized immunoglobulincomprising the heavy chain set forth in SEQ ID NO: 47. In anotherembodiment, the invention features a humanized immunoglobulin comprisingthe heavy chain set forth in SEQ ID NO: 48. In still another embodiment,the invention features a humanized immunoglobulin comprising the lightchain set forth in SEQ ID NO: 49. In yet another embodiment, theinvention features a humanized immunoglobulin comprising the light chainset forth in SEQ ID NO: 50.

In one embodiment, the invention features a heavy chain comprising acomplementarity determining region (CDR) and at least one variableregion framework residue from the monoclonal antibody 11K2 heavy chainset forth as SEQ ID NO: 27, wherein the residue is selected from thegroup consisting of L27, I29, and T73 (Kabat numbering convention), andwherein the remainder of the heavy chain is from a human immunoglobulin.In one embodiment, the heavy chain comprises variable region frameworkresidues L27, I29, and T73. In another embodiment, the heavy chainfurther comprises at least one variable region framework residueselected from the group consisting of N28, K30, I48, and A67 (Kabatnumbering convention). In still another embodiment of the invention, theheavy chain comprises variable region framework residues N28, K30, I48,and A67.

In another embodiment, the invention features a light chain comprising acomplementarity determining region (CDR) and at least one variableregion framework residue from the monoclonal antibody 11K2 light chainset forth as SEQ ID NO: 28, wherein the residue is selected from thegroup consisting of S49 and Y71 (Kabat numbering convention), andwherein the remainder of the light chain is from a human immunoglobulin.In one embodiment, the light chain comprises variable region frameworkresidues S49 and Y71. In another embodiment, the light chain furthercomprises variable region framework residue K69 (Kabat numberingconvention).

In another embodiment, the invention features a humanized immunoglobulinor antigen-binding fragment thereof comprising heavy chain complementarydetermining regions as set forth in SEQ ID NO: 29, SEQ ID NO: 30, andSEQ ID NO: 31, and at least one variable region framework residue fromthe monoclonal antibody 11K2 heavy chain set forth as SEQ ID NO: 27,wherein the residue is selected from the group consisting of L27, N28,I29, K30, I48, A67, and T73 (Kabat numbering). In one embodiment, theheavy chain comprises variable region framework residues L27, N28, I29,K30, I48, A67, and T73. In another embodiment, the heavy chain comprisesvariable region framework residues L27, I29, and T73.

In yet another embodiment, the invention features a humanizedimmunoglobulin or antigen-binding fragment thereof comprising lightchain complementary determining regions as set forth in SEQ ID NO: 32,SEQ ID NO: 33, and SEQ ID NO: 34, and at least one variable regionframework residues from the monoclonal antibody 11K2 light chain setforth as SEQ ID NO: 28, wherein the residue is selected from the groupconsisting of S49, K69, and Y71 (Kabat numbering). In one embodiment,the light chain comprises variable region framework residues S49, K69,and Y71. In another embodiment, the light chain further comprisesvariable region framework residues S49 and Y71.

In still another embodiment, the invention describes a humanizedimmunoglobulin or antigen-binding fragment comprising the light chain ofthe invention and the heavy chain of the invention.

In one embodiment, the invention features a humanized immunoglobulin orantigen-binding fragment comprising

-   -   a) a heavy chain comprising a complementarity determining region        (CDR) and at least one variable region framework residue from        the monoclonal antibody 11K2 heavy chain set forth as SEQ ID NO:        27, wherein the residue is selected from the group consisting of        L27, N28, I29, K30, I48, A67 and T73 (Kabat numbering        convention), and    -   b) a light chain comprising a complementarity determining region        (CDR) and at least one variable region framework residue from        the monoclonal antibody 11K2 light chain set forth as SEQ ID NO:        28, wherein the residue is selected from the group consisting of        S49, K69, and Y71 (Kabat numbering convention),        wherein the remainder of the heavy and light chains are from a        human immunoglobulin. In a certain embodiment, the heavy chain        comprises variable region framework residues L27, I29, and T73,        and the light chain comprises variable region framework residues        S49 and Y71. In another embodiment, the heavy chain comprises        variable region framework residues L27, N28, I29, K30, I48, A67        and T73, and the light chain comprises variable region framework        residues S49, K69, and Y71. In yet another embodiment, the heavy        chain comprises variable region framework residues L27, I29, and        T73, and the light chain comprises variable region framework        residues S49, K69, and Y71. In yet a further embodiment, the        heavy chain comprises variable region framework residues L27,        N28, I29, K30, I48, A67 and T73, and the light chain comprises        variable region framework residues S49 and Y71.

In one embodiment, the invention features a humanized immunoglobulin orantigen-binding fragment comprising

a) heavy chain complementary determining regions as set forth in SEQ IDNO: 29, SEQ ID NO: 30, and SEQ ID NO: 31, and variable region frameworkresidues L27, N28, I29, K30, I48, A67, and T73 (Kabat numbering) fromthe monoclonal antibody 11K2 heavy chain set forth as SEQ ID NO: 27, and

b) light chain complementary determining regions as set forth in SEQ IDNO: 32, SEQ ID NO: 33, and SEQ ID NO: 34, and variable region frameworkresidues S49, K69, and Y71 (Kabat numbering), from the monoclonalantibody 11K2 light chain set forth as SEQ ID NO: 28, wherein theremainder of the heavy and light chains are from a human immunoglobulin.In one embodiment of the invention, the immunoglobulin is modified byreducing or eliminating at least one potential glycosylation site.

In one embodiment, the immunoglobulin or antigen-binding fragment of theinvention, binds to MCP-1. In one embodiment, the immunoglobulin orantigen-binding fragment of the invention, specifically binds to MCP-1with a binding affinity of at least 10⁻⁹ M. In another embodiment, theimmunoglobulin or antigen-binding fragment of the invention,specifically binds to MCP-1 with a binding affinity of at least 10⁻¹⁰ M.In still another embodiment, the immunoglobulin or antigen-bindingfragment of the invention specifically binds to MCP-1 with a bindingaffinity of at least 10⁻¹¹ M. In still another embodiment, theimmunoglobulin or antigen-binding fragment of the invention, furtherbinds to MCP-2 with a binding affinity of at least 10⁻⁷ M. In anotherembodiment, the immunoglobulin or antigen-binding fragment of theinvention, further binds to MCP-2 with a binding affinity of at least10⁻⁸ M. In yet another embodiment, the immunoglobulin or antigen-bindingfragment of the invention, further binds to MCP-2 with a bindingaffinity of at least 10⁻⁹ M.

In one embodiment, the immunoglobulin or antigen-binding fragment of theinvention binds to MCP-2. In one embodiment, the immunoglobulin orantigen-binding fragment of the invention specifically binds to MCP-2with a binding affinity of at least 10⁻⁷ M. In yet another embodiment,the immunoglobulin or antigen-binding fragment of the inventionspecifically binds to MCP-2 with a binding affinity of at least 10⁻⁸ M.In yet another embodiment, the immunoglobulin or antigen-bindingfragment of the invention specifically binds to MCP-2 with a bindingaffinity of at least 10⁻⁹ M.

In one embodiment, the immunoglobulin or antigen-binding fragment of theinvention binds to MCP-1 and MCP-2. In another embodiment, theimmunoglobulin or antigen-binding fragment of the invention binds to anepitope within MCP-1, MCP-2, and MCP-3.

Another embodiment of the invention features a method of treating adisorder associated with detrimental MCP activity comprisingadministering to a subject having said disorder, a nucleic acid moleculethat encodes an immunoglobulin heavy chain comprising the amino acidsequence of SEQ ID NO: 53 or the amino acid sequence of SEQ ID NO: 54and a nucleic acid molecule that encodes an immunoglobulin light chainthat comprises the amino acid sequence of SEQ ID NO: 55 or the aminoacid sequence of SEQ ID NO: 56, under conditions such that saidimmunoglobulin chains are expressed, such that a beneficial therapeuticresponse in said subject is generated.

In one embodiment, the invention features an antibody comprising thesame heavy and light chain polypeptide sequences as an antibody producedby a CHO cell line secreting humanized 11K2 (version H2L1) clone 3F2(ATCC patent deposit designation PTA-5308).

In another embodiment, the invention describes an isolated nucleic acidmolecule encoding the heavy chain of the immunoglobulin orantigen-binding fragment of the invention. In another embodiment, theinvention features an isolated nucleic acid molecule encoding the lightchain of immunoglobulin or antigen-binding fragment of the invention. Instill another embodiment, the invention features an isolated nucleicacid molecule encoding the immunoglobulin or antigen-binding fragment ofthe invention.

In one embodiment, the invention features an isolated nucleic acidmolecule comprising a nucleotide sequence corresponding to the aminoacid sequence selected from the group consisting of SEQ ID NO: 47, SEQID NO: 48, SEQ ID NO: 49, and SEQ ID NO: 50.

In still another embodiment, the invention features an isolated nucleicacid comprising a coding sequence for the heavy chain of an antibodyproduced by a CHO cell line secreting humanized 11K2 (version H2L1)clone 3F2 (ATCC patent deposit designation PTA-5308). In anotherembodiment, the invention features an isolated nucleic acid comprising acoding sequence for the light chain of an antibody produced by a CHOcell line secreting humanized 11K2 (version H2L1) clone 3F2 (ATCC patentdeposit designation PTA-5308).

In another embodiment, the invention features a cell line of humanized11K2 (version H2L1) clone 3F2 (ATCC patent deposit designationPTA-5308).

One embodiment of the invention features a humanized immunoglobulinheavy chain or antigen-binding portion thereof comprising variableregion complementary determining regions (CDRs) from the 1A1immunoglobulin heavy chain variable region sequence set forth as SEQ IDNO: 11, and variable framework regions from a human acceptorimmunoglobulin heavy chain sequence, provided that at least oneframework residue is substituted with the corresponding amino acidresidue from the mouse 1A1 heavy chain variable region sequence, whereinthe framework residue is selected from the group consisting of:

a residue that non-covalently binds antigen directly;

a residue adjacent to a CDR;

a CDR-interacting residue; and

a residue participating in the VL-VH interface.

The invention features a humanized immunoglobulin heavy chain orantigen-binding portion thereof, wherein a CDR-interacting residue isidentified by modeling the 1A1 heavy chain based on the solved structureof a murine immunoglobulin heavy chain that shares at least 70% sequenceidentity with the 1A1 heavy chain. The invention also features ahumanized immunoglobulin heavy chain or antigen-binding portion thereof,wherein a CDR-interacting residue is identified by modeling the 1A1heavy chain based on the solved structure of a murine immunoglobulinheavy chain that shares at least 80% sequence identity with the 1A1heavy chain. The invention further features a humanized immunoglobulinheavy chain or antigen-binding portion thereof, wherein aCDR-interacting residue is identified by modeling the 1A1 heavy chainbased on the solved structure of a murine immunoglobulin heavy chainthat shares at least 90% sequence identity with the 1A1 heavy chain.

In another embodiment, the invention features a humanized immunoglobulinlight chain or antigen-binding portion thereof comprising variableregion complementary determining regions (CDRs) from the 1A1immunoglobulin light chain variable region sequence set forth as SEQ IDNO: 12 and variable framework regions from a human acceptorimmunoglobulin light chain, provided that at least one framework residueis to substituted with the corresponding amino acid residue from themouse 1A1 light chain variable region sequence, wherein the frameworkresidue is selected from the group consisting of:

a residue that non-covalently binds antigen directly;

a residue adjacent to a CDR;

a CDR-interacting residue; and

a residue participating in the VL-VH interface.

The invention features a humanized immunoglobulin light chain orantigen-binding portion thereof, wherein a CDR-interacting residue isidentified by modeling the 1A1 light chain based on the solved structureof a murine immunoglobulin light chain that shares at least 70% sequenceidentity with the 1A1 light chain. The invention also features ahumanized immunoglobulin light chain, wherein a CDR-interacting residueis identified by modeling the 1A1 light chain based on the solvedstructure of a murine immunoglobulin light chain that shares at least80% sequence identity with the 1A1 light chain. The invention features ahumanized immunoglobulin light chain, wherein a CDR-interacting residueis identified by modeling the 1A1 light chain based on the solvedstructure of a murine immunoglobulin light chain that shares at least90% sequence identity with the 1A1 light chain.

In another embodiment, the invention features a humanized immunoglobulinheavy chain or antigen-binding portion thereof comprising variableregion complementary determining regions (CDRs) from the 1A1immunoglobulin heavy chain variable region sequence set forth as SEQ IDNO: 11, and variable framework regions from a human acceptorimmunoglobulin heavy chain sequence, provided that at least oneframework residue is substituted with the corresponding amino acidresidue from the mouse 1A1 heavy chain variable region sequence, whereinthe framework residue is a residue capable of affecting heavy chainvariable region conformation or function as identified by analysis of athree-dimensional model of the variable region. 70. The invention alsofeatures a humanized immunoglobulin of a heavy chain or antigen-bindingportion thereof, wherein the framework residue is selected from thegroup consisting of a residue capable of interacting with antigen, aresidue proximal to the antigen binding site, a residue capable ofinteracting with a CDR, a residue adjacent to a CDR, a residue within 6Å of a CDR residue, a canonical residue, a vernier zone residue, aninterchain packing residue, and a rare residue.

In another embodiment, the invention features a humanized immunoglobulinlight chain or antigen-binding portion thereof, comprising variableregion complementary determining regions (CDRs) from the 1A1immunoglobulin light chain variable region sequence set forth as SEQ IDNO: 12, and variable framework regions from a human acceptorimmunoglobulin light chain, provided that at least one framework residueis substituted with the corresponding amino acid residue from the mouse1A1 light chain variable region sequence, wherein the framework residueis a residue capable of affecting light chain variable regionconformation or function as identified by analysis of athree-dimensional model of the variable region. The invention alsofeatures a humanized immunoglobulin of a light chain or antigen-bindingportion thereof, wherein the framework residue is selected from thegroup consisting of a residue capable of interacting with antigen, aresidue proximal to the antigen binding site, a residue capable ofinteracting with a CDR, a residue adjacent to a CDR, a residue within 6Å of a CDR residue, a canonical residue, a vernier zone residue, aninterchain packing residue, and an unusual residue.

In one embodiment the invention features a heavy and/or light chain,wherein the framework residue is identified by modeling the 1A1 heavychain based on the solved structure of a murine immunoglobulin heavychain that shares at least 70% sequence identity with the 1A1 heavyand/or light chain. In another embodiment the invention features a heavyand/or light chain, wherein the framework residue is identified bymodeling the 1A1 heavy or light chain based on the solved structure of amurine immunoglobulin heavy and/or light chain that shares at least 80%sequence identity with the 1A1 heavy or light chain. In anotherembodiment the invention features a heavy and/or light chain, whereinthe framework residue is identified by modeling the 1A1 heavy chainbased on the solved structure of a murine immunoglobulin heavy and/orlight chain that shares at least 90% sequence identity with the 1A1heavy or light chain.

In still another embodiment, the invention features a heavy chaincomprising the complementarity determining regions (CDRs) and variableregion framework residue H29, H30, H73, H91, H93, and H94 (Kabatnumbering convention) from the monoclonal antibody 1A1 heavy chain,wherein the remainder of the heavy chain is from a human immunoglobulin.In another embodiment, the heavy chain of the invention furthercomprises at least one variable framework residue from the monoclonalantibody 1A1 heavy chain selected from the group consisting of H27, H28,H66, H69, and H76 (Kabat numbering convention).

In still another embodiment, the invention includes a light chaincomprising the complementarity determining regions (CDRs) and variableframework residues L2 and L36 (Kabat numbering convention) from themonoclonal antibody 1A1 light chain, wherein the remainder of the lightchain is from a human immunoglobulin. The invention also features alight chain further comprising the variable framework residue from themonoclonal antibody 1A1 heavy chain L45 (Kabat numbering convention).

In one embodiment, the invention features a humanized immunoglobulincomprising the heavy chain set forth in SEQ ID NO: 53. In still anotherembodiment, the invention includes a humanized immunoglobulin comprisingthe heavy chain set forth in SEQ ID NO: 54. In a further embodiment, theinvention features a humanized immunoglobulin comprising the light chainset forth in SEQ ID NO: 55. The invention also features a humanizedimmunoglobulin comprising the light chain set forth in SEQ ID NO: 56.The invention also features a humanized immunoglobulin comprising aheavy chain comprising SEQ ID NO: 53 or SEQ ID NO: 54 and a light chaincomprising SEQ ID NO: 55 or SEQ ID NO: 56.

In one embodiment, the invention features an immunoglobulin or antigenbinding fragment, which specifically binds to MCP-1 with a bindingaffinity of at least 10⁻⁹ M. In another embodiment, the immunoglobulinor antigen binding fragment of the invention specifically binds to MCP-1with a binding affinity of at least 10⁻¹⁰ M. In still a furtherembodiment, the immunoglobulin or antigen binding fragment of theinvention specifically binds to MCP-1 with a binding affinity of atleast 10⁻¹¹ M. In one embodiment, the immunoglobulin or antigen-bindingfragment of the invention binds to MCP-2. In another embodiment, theimmunoglobulin or antigen-binding fragment of the invention binds toMCP-1 and MCP-2. In still another embodiment, the immunoglobulin orantigen-binding fragment of the invention binds to an epitope withinMCP-1, MCP-2, and MCP-3.

In still another embodiment, the invention features a method foridentifying residues amenable to substitution in a humanized 1A1immunoglobulin variable framework region, comprising modeling thethree-dimensional structure of the 1A1 variable region based on a solvedimmunoglobulin structure and analyzing said model for residues capableof affecting 1A1 immunoglobulin variable region conformation or functionsuch that residues amenable to substitution are identified. Theinvention also includes use of the variable region sequence set forth asSEQ ID NO: 11 or SEQ ID NO: 12, or any, portion thereof, in producing athree-dimensional image of a 1A1 immunoglobulin, 1A1 immunoglobulinchain, or domain thereof.

In one embodiment, the immunoglobulin or antigen-binding fragment of theinvention is modified by reducing or eliminating at least one potentialglycosylation site. In another embodiment, the immunoglobulin orantigen-binding fragment of the invention is modified by conjugation toa carrier selected from polyethylene glycol and albumen. In yet anotherembodiment, the immunoglobulin or antigen-binding fragment of theinvention is modified to reduce at least one constant region-mediatedbiological effector function relative to an unmodified antibody.

In one embodiment, the heavy chain isotype of the immunoglobulin orantigen-binding fragment of the invention is gamma 1.

In one embodiment, the fragment of the invention is a Fab fragment.

In one embodiment, the invention features an immunoglobulin orantigen-binding fragment which inhibits MCP-induced chemotaxis. In acertain embodiment, the immunoglobulin or antigen-binding fragment ofthe invention inhibits MCP-1-induced chemotaxis, MCP-2-inducedchemotaxis, or both MCP-1-induced and MCP-2-induced chemotaxis.

In one embodiment, the immunoglobulin or antigen-binding fragment of theinvention inhibits MCP-induced collagen expression. In anotherembodiment, the invention features an immunoglobulin or antigen-bindingfragment, wherein the immunoglobulin or antigen-binding fragmentinhibits MCP-1-induced collagen expression, MCP-2-induced collagenexpression, or both MCP-1-induced and MCP-2-induced collagen expression.

In one embodiment, the invention features an immunoglobulin orantigen-binding fragment which inhibits MCP-1-induced angiogenesis. Incertain embodiments, an immunoglobulin or antigen-binding fragment ofthe invention inhibits MCP-1-induced angiogenesis, MCP-2-inducedangiogenesis, or both MCP-1-induced and MCP-2-induced angiogenesis.

In one embodiment, the immunoglobulin or antigen-binding fragment of anythe invention reduces inflammation in a subject. In one embodiment, theinflammation is associated with a disorder selected from the groupconsisting of arthritis, multiple sclerosis, cirrhosis, atherosclerosis,and breast carcinoma.

In one embodiment, an immunoglobulin or antigen-binding fragment of theinvention reduces fibrosis in a subject.

In one embodiment, the invention features a pharmaceutical compositioncomprising the immunoglobulin or antigen-binding fragment of theinvention and a pharmaceutical carrier.

In another embodiment, the invention features a host cell comprising thenucleic acid molecule of a immunoglobulin or antigen-binding fragment ofthe invention. In one embodiment, the host cell of the invention ismammalian. In another embodiment, the host cell of the invention isbacterial. In still another embodiment, the invention describes a methodof producing an antibody or antigen binding fragment thereof of theinvention, comprising culturing the host cell under conditions such thatthe antibody or fragment is produced and isolating said antibody fromthe host cell or culture.

In still another embodiment, the invention provides a method ofpreventing or treating a disorder associated with detrimental MCPactivity in a subject, comprising administering to the subject aneffective amount of an immunoglobulin or antigen binding fragment of theinvention. In one embodiment, the effective amount of immunoglobulin orantigen binding fragment thereof is 1-10 mg/kg body weight. In anotherembodiment, the disorder is selected from the group consisting ofglomerulonephritis, scleroderma, cirrhosis, multiple sclerosis, lupusnephritis, atherosclerosis, inflammatory bowel diseases or rheumatoidarthritis.

In yet another embodiment, the invention provides a method of preventingor treating MCP-associated inflammation in a subject, comprisingadministering to the subject an effective amount of an immunoglobulin orantigen binding fragment of the invention.

In yet another embodiment of the invention, a method of preventing ortreating MCP-associated inflammation in a subject is described,comprising administering to the subject an effective amount of thehumanized immunoglobulin or antigen-binding portion of the invention.Effective amounts of humanized immunoglobulin or antigen-binding portiondescribed in the invention include, for to example, 1 mg/kg body weightto 10 mg/kg body weight.

In yet another embodiment, the invention provides a method of preventingor treating a fibrotic disorder in a subject comprising administering tothe subject an effective amount of an immunoglobulin or antigen bindingfragment of the invention.

In yet another embodiment, the invention provides a method of preventingor treating cancer in a subject comprising administering to the subjectan effective amount of an immunoglobulin or antigen binding fragment ofthe invention.

In yet another embodiment, the invention provides a method of preventingor treating an immunopathologic disorder comprising administering to thesubject an effective amount of an immunoglobulin or antigen bindingfragment of the invention.

Another embodiment of the invention features use of the antibodies orantigen-binding fragments of the invention for preventing or treating aninflammatory disorder, e.g., Alzheimer's, severe asthma, atopicdermatitis, cachexia, CHF-ischemia, coronary restinosis, Crohn'sdisease, diabetic nephropathy, lymphoma, psoriasis,fibrosis/radiation-induced, juvenile arthritis, stroke, inflammation ofthe brain or central nervous system caused by trauma, and ulcerativecolitis, inflammation due to corneal transplantation, chronicobstructive pulmonary disease, hepatitis C, multiple myeloma, andosteoarthritis.

Yet another embodiment of the invention features used of the antibodiesor antigen-binding fragments of the invention for preventing or treatinga neurodegenerative disorder. Neurodegenerative disorders which can betreated by the antibodies or antigen-binding fragments thereof, include,but are not limited to, Alzheimer's, stroke, traumatic brain or centralnervous system injuries, ALS/motor neuron disease, diabetic peripheralneuropathy, diabetic retinopathy, Huntington's disease, maculardegeneration, and Parkinson's disease.

In yet another embodiment, the invention includes a heavy chain of ananti-MCP antibody which contacts residues R30, T32, S34, K38, E39, V41,P55, K56, Q61, M64 of MCP-1. The invention further includes a lightchain anti-MCP antibody which contacts residues D65, D68, K69 of MCP-1.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 graphically depicts results of a chemotaxis assay using purified11K2, 1A1, D9, and 2O24 to inhibit chemotaxis in response to MCP-1,MCP-2, and a combination of MCP-1/MCP-2. The results show thatchemotaxis to a combination of MCP-1 and MCP-2 is inhibited by 11K2 and1A1.

FIG. 2 graphically depicts the results of a chemotaxis assay of cells inresponse to MCP-1. FIG. 2A graphically depicts results using monoclonalantibodies 5D3-F7 (BD Biosciences, Pharmingen, San Diego, Calif.), 1M11,3N10, 5J23, and 11K2 in response to 20 ng/mL of MCP-1. FIG. 2Bgraphically depicts results using 20 ng/mL of murine MCP-1 (JE) andmonoclonal antibodies 2H5 (BD Biosciences, Pharmingen, San Diego,Calif.), 1M11, 3N10, 5J23, and 11K2.

FIG. 3 graphically depicts results of a chemotaxis assay whichdemonstrates that monocyte chemotaxis mediated by cytokines secretedfrom stimulated rheumatoid arthritis (RA) fibroblasts is inhibited bypan-MCP mAbs (1A1 and 11K2) and MCP-1 mAb D9.

FIG. 4 graphically depicts results from a calcium flux assay using 11K2(mAb and Fab fragments thereof) at various concentrations, includingnone (FIG. 4A), 20 nM mAb (FIG. 4B), and 60 nM Fab (FIG. 4C).

FIG. 5 graphically depicts results from a chemotaxis assay using pan-MCPantibodies demonstrating pan-MCP antibodies 11K2 and 1A1 increase MCP-2mediated chemotaxis at low mAb concentrations (FIG. 5A). Blocking isalso observed with MCP-2 mAb 281 (RD Systems, Minneapolis, Minn.). FIG.5B graphically depicts a chemotaxis assay using the pan-MCP mAb 11K2 andthe Fab fragment of 11K2.

FIG. 6 graphically depicts results from a MCP-2 calcium flux assay whichdepicts results from 55.5 nM of MCP-2 alone, depicts results whichdemonstrate that the 11K2 monoclonal antibody shows agonistic activity(6B), and, as compared to 55.5 nM of MCP-2 alone (6A), FIGS. 6C and 6Ddepict results which demonstrate that Fab and F(ab′)2 fragments of 11K2are inhibitory in this assay.

FIG. 7 shows the amino acid and nucleotide sequences of the variableheavy region of the murine version of the 1A1 antibody (7A), as well asthe amino acid and nucleotide sequences of the 1A1 variable light region(7B). CDR regions are underlined.

FIG. 8 shows the amino acid and nucleotide sequences of the murine 11K2variable heavy region (FIG. 8A), and the amino acid and nucleotidesequences of murine 11K2 variable light region (FIG. 8B). CDR regionsare underlined.

FIG. 9 shows the nucleotide and amino acid sequences of a heavy chainchimera (variable and constant regions) of the 11K2 antibody (9A). Thevariable region is set forth at nucleotides 1-351 (amino acids 1-117) ofthe heavy chain. FIG. 9B shows the DNA/amino acid comparison of the 11K2light chain chimera (variable and constant regions). The variable regionis set forth at nucleotides 1-321 (amino acids 1-107) of the lightchain. All CDRs are underlined.

FIG. 10 shows the nucleotide and amino acid sequences of the light andheavy chains of humanized 11K2 antibody. FIG. 10A shows the sequence ofhumanized version 1 including heavy chain variable and constant regions.FIG. 10B shows the sequence of humanized version 2 including heavy chainvariable and constant regions. FIG. 10C shows the sequence of humanizedversion 1 light chain variable and constant regions. FIG. 10D shows thesequence of humanized version 2 light chain variable and constantregions. All CDR regions are underlined, and all backmutations arehighlighted in bold.

FIG. 11 shows an alignment of the murine 11K2 antibody and the humanized11K2 (versions 1 and 2) for the variable heavy chain region (A) and thevariable light chain region (B). All CDRs are underlined and in bold.

FIG. 12 graphically depicts results from a neutralization assay usingmAb 11K2, chimeric 11K2, aglycosylated chimeric 11K2, H1/L1 humanized11K2, H2/L2 humanized 11K2, H1/L2 humanized 11K2, and H2/L1 humanized11K2 antibodies (whole and Fab fragments). FIG. 12A graphically depictsresults from a neutralization assay using 2.3 nM MCP-1. FIG. 12Bgraphically depicts results from a neutralization assay using 56 nMMCP-2. All of the antibodies tested exhibit an agonist activity forMCP-2 at low concentrations.

FIG. 13 graphically depicts results from an experiment whichdemonstrates that PEGylation of 11K2 Fab retains in vitro activity.

FIG. 14 graphically depicts results from an ELISA experiment, which showreactivity of the humanized and chimeric 11K2 antibodies with huMCP-1,primate MCP-1, and muMCP-1. FIG. 14B graphically depicts results from anELISA experiment, which show the reactivity of humanized and chimeric11K2 antibodies with MCP-2.

FIG. 15 graphically depicts the therapeutic effect of mouse monoclonalantibody 11K2 treatment on survival of mice afflicted by TNBS-inducedcolitis.

FIG. 16 graphically depicts the reduction of MCP-1 levels seen inTNBS-induced colitis mice treated with monoclonal antibody 11K2.

FIG. 17 graphically depicts the results of experiments demonstrating theefficacy of hu11K2 and pegylated 11K2-Fab (11K2 PEG-Fab) to inhibitcolitis in a TNBS-induced mice.

FIG. 18 graphically depicts the therapeutic effect of monoclonalantibody 11K2 administration to TNBS-induced mice. At day 7post-TNBS-induction 11K2 treatment resulted in elevated body weight,reduced MCP and TNFα levels; and inhibition of myeloperoxidase (MPO)activity, as compared with untreated and control mAb-treated colitismodel mice.

FIG. 19 graphically depicts results demonstrating a reduction inatherosclerotic plaque size (total plaque area) in apoE-deficient micetreated with mouse monoclonal antibody 11K2.

DETAILED DESCRIPTION OF THE INVENTION

The present invention features new immunological reagents and methodsfor preventing or treating disorders associated with detrimental MCPactivity. The invention is based, at least in part, on thecharacterization of two monoclonal immunoglobulins, 11K2 and 1A1,effective at binding MCPs (Aβ) (e.g., MCP-1, MCP-2, and MCP-3). Theinvention is further based on the determination and structuralcharacterization of the primary and secondary structure of the variablelight and heavy chains of these immunoglobulins and the identificationof residues important for activity and immunogenicity.

Immunoglobulins are featured which include a variable light and/orvariable heavy chain of the preferred monoclonal immunoglobulinsdescribed herein. Preferred immunoglobulins, e.g., therapeuticimmunoglobulins, are featured which include a humanized variable lightand/or humanized variable heavy chain. Preferred variable light and/orvariable heavy chains include a complementarity determining region (CDR)from the monoclonal immunoglobulin (e.g., donor immunoglobulin) andvariable framework regions substantially from a human acceptorimmunoglobulin. The phrase “substantially from a human acceptorimmunoglobulin” means that the majority or key framework residues arefrom the human acceptor sequence, allowing however, for substitution ofresidues at certain positions with residues selected to improve activityof the humanized immunoglobulin (e.g., alter activity such that it moreclosely mimics the activity of the donor immunoglobulin) or selected todecrease the immunogenicity of the humanized immunoglobulin.

In one embodiment, the invention features a humanized immunoglobulinlight or heavy chain that includes 11K2 variable region complementaritydetermining regions (CDRs) (i.e., includes one, two or three CDRs fromthe light chain variable region sequence set forth as SEQ ID NO: 28 orincludes one, two or three CDRs from the heavy chain variable regionsequence set forth as SEQ ID NO: 27), and includes a variable frameworkregion substantially from a human acceptor immunoglobulin light or heavychain sequence, provided that at least one residue of the frameworkresidue is backmutated to a corresponding marine residue, wherein saidbackmutation does not substantially affect the ability of the chain todirect MCP binding.

In another embodiment, the invention features a humanized immunoglobulinlight or heavy chain that includes 11K2 variable region complementaritydetermining regions (CDRs) (e.g., includes one, two or three CDRs fromthe light chain variable region sequence set forth as SEQ ID NO: 28and/or includes one, two or three CDRs from the heavy chain variableregion sequence set forth as SEQ ID NO: 27), and includes a variableframework region substantially from a human acceptor immunoglobulinlight or heavy chain sequence, provided that at least one frameworkresidue is substituted with the corresponding amino acid residue fromthe mouse 11K2 light or heavy chain variable region sequence, where theframework residue is selected from the group consisting of (a) a residuethat non-covalently binds antigen directly; (b) a residue adjacent to aCDR; (c) a CDR-interacting residue (e.g., identified by modeling thelight or heavy chain on the solved structure of a homologous knownimmunoglobulin chain); and (d) a residue participating in the VL-VHinterface.

In another embodiment, the invention features a humanized immunoglobulinlight or heavy chain that includes 11K2 variable region CDRs andvariable framework regions from a human acceptor immunoglobulin light orheavy chain sequence, provided that at least one framework residue issubstituted with the corresponding amino acid residue from the mouse11K2 light or heavy chain variable region sequence, where the frameworkresidue is a residue capable of affecting light chain variable regionconformation or function as identified by analysis of athree-dimensional model of the variable region, for example a residuecapable of interacting with antigen, a residue proximal to the antigenbinding site, a residue capable of interacting with a CDR, a residueadjacent to a CDR, a residue within 6 Å of a CDR residue, a canonicalresidue, a vernier zone residue, an interchain packing residue, or anunusual residue.

In another embodiment, the invention features a humanized immunoglobulinlight chain that includes 11K2 variable region CDRs (e.g., from the 11K2light chain variable region sequence set forth as SEQ ID NO: 28), andincludes a human acceptor immunoglobulin variable framework region,provided that at least one framework residue selected from the groupconsisting of L49, L69 and L71 (Kabat numbering convention) issubstituted with the corresponding amino acid residue from the mouse11K2 light chain variable region sequence. In another embodiment, theinvention features a humanized immunoglobulin heavy chain that includes11K2 variable region CDRs (e.g., from the 11K2 heavy chain variableregion sequence set forth as SEQ ID NO: 27), and includes a humanacceptor immunoglobulin variable framework region, provided that atleast one framework residue selected from the group consisting of H27,H28, H29, H30, H48, H67, and H73 (Kabat numbering convention) issubstituted with the corresponding amino acid residue from the mouse11K2 heavy chain variable region sequence.

Preferred light chains include framework regions of the subtype kappa 1(Kabat convention), for example, framework regions from the acceptorimmunoglobulin GI-486875. Preferred heavy chains include frameworkregions of the subtype 1 (Kabat convention), for example, frameworkregions from the acceptor immunoglobulin Kabat ID 000054.

In one embodiment, the invention features a humanized immunoglobulinlight or heavy chain that includes 1A1 variable region complementaritydetermining regions (CDRs) (i.e., includes one, two or three CDRs fromthe light chain variable region sequence set forth as SEQ ID NO: 12 orincludes one, two or three CDRs from the heavy chain variable regionsequence set forth as SEQ ID NO: 11), and includes a variable frameworkregion substantially from a human acceptor immunoglobulin light or heavychain sequence, provided that at least one residue of the frameworkresidue is backmutated to a corresponding murine residue, wherein saidbackmutation does not substantially affect the ability of the chain todirect MCP binding.

In another embodiment, the invention features a humanized immunoglobulinlight or heavy chain that includes 1A1 variable region complementaritydetermining regions (CDRs) (e.g., includes one, two or three CDRs fromthe light chain variable region sequence set forth as SEQ ID NO: 12and/or includes one, two or three CDRs from the heavy chain variableregion sequence set forth as SEQ ID NO: 11), and includes a variableframework region substantially from a human acceptor immunoglobulinlight or heavy chain sequence, provided that at least one frameworkresidue is substituted with the corresponding amino acid residue fromthe mouse 1A1 light or heavy chain variable region sequence, where theframework residue is selected from the group consisting of (a) a residuethat non-covalently binds antigen directly; (b) a residue adjacent to aCDR; (c) a CDR-interacting residue (e.g., identified by modeling thelight or heavy chain on the solved structure of a homologous knownimmunoglobulin chain); and (d) a residue participating in the VL-VHinterface.

In another embodiment, the invention features a humanized immunoglobulinlight or heavy chain that includes 1A1 variable region CDRs and variableframework regions from a human acceptor immunoglobulin light or heavychain sequence, provided that at least one framework residue issubstituted with the corresponding amino acid residue from the mouse 1A1light or heavy chain variable region sequence, where the frameworkresidue is a residue capable of affecting light chain variable regionconformation or function as identified by analysis of athree-dimensional model of the variable region, for example a residuecapable of interacting with antigen, a residue proximal to the antigenbinding site, a residue capable of interacting with a CDR, a residueadjacent to a CDR, a residue within 6 Å of a CDR residue, a canonicalresidue, a vernier zone residue, an interchain packing residue, or anunusual residue.

In another embodiment, the invention features a humanized immunoglobulinthat includes a light chain and a heavy chain, as described above, or anantigen-binding fragment of said immunoglobulin. In an exemplaryembodiment, the humanized immunoglobulin binds (e.g., specificallybinds) to MCP-1 with a binding affinity of at least 10⁷ M⁻¹, 10⁸ M⁻¹, or10⁹ M⁻¹.

In another embodiment, the invention features chimeric immunoglobulinsthat include 11K2 variable regions (e.g., the variable region sequencesset forth as SEQ ID NO: 27 or SEQ ID NO: 28). In yet another embodiment,the invention features an immunoglobulin, or antigen-binding fragmentthereof, including a variable heavy chain region as set forth in SEQ IDNO: 47 or SEQ ID NO: 48 and a variable light chain region as set forthin SEQ ID NO: 49 or SEQ ID NO: 50.

In another embodiment, the invention features chimeric immunoglobulinsthat include 1A1 variable regions (e.g., the variable region sequencesset forth as SEQ ID NO: 11 or SEQ ID NO: 12). In yet another embodiment,the invention features an immunoglobulin, or antigen-binding fragmentthereof including a variable heavy chain region as set forth in SEQ IDNO: 53 or SEQ ID NO: 54 and a variable light chain region as set forthin SEQ ID NO: 55 or SEQ ID NO: 56. In yet another embodiment, theimmunoglobulin, or antigen-binding fragment thereof, further includesconstant regions from IgG1.

The immunoglobulins described herein are particularly suited for use intherapeutic methods aimed at preventing or treating disorders associatedwith detrimental MCP activity. In one embodiment, the invention featuresa method of preventing or treating a disorder associated withdetrimental MCP activity that involves administering to the subject aneffective dosage of a humanized immunoglobulin as described herein. Inanother embodiment, the invention features pharmaceutical compositionsthat include a humanized immunoglobulin as described herein and apharmaceutical carrier. Also featured are isolated nucleic acidmolecules, vectors and host cells for producing the immunoglobulins orimmunoglobulin fragments or chains described herein, as well as methodsfor producing said immunoglobulins, immunoglobulin fragments orimmunoglobulin chains

The present invention further features a method for identifying 1A1 or11K2 residues amenable to substitution when producing a humanized 1A1 or11K2 immunoglobulin, respectively. For example, a method for identifyingvariable framework region residues amenable to substitution involvesmodeling the three-dimensional structure of the 1A1 or 11K2 variableregion on a solved homologous immunoglobulin structure and analyzingsaid model for residues capable of affecting 1A1 or 11K2 immunoglobulinvariable region conformation or function, such that residues amenable tosubstitution are identified. The invention further features use of thevariable region sequence set forth as SEQ ID NO: 27 or SEQ ID NO: 28, orany portion thereof, in producing a three-dimensional image of a 11K2immunoglobulin, 11K2 immunoglobulin chain, or domain thereof. Alsofeatured is the use of the variable region sequence set forth as SEQ NO:11, or SEQ ID NO: 12, or any portion thereof, in producing athree-dimensional image of a 1A1 immunoglobulin, 1A1 immunoglobulinchain, or domain thereof.

Prior to describing the invention, it may be helpful to an understandingthereof to set forth definitions of certain terms to be usedhereinafter.

The term “immunoglobulin” or “antibody” (used interchangeably herein)refers to an antigen-binding protein having a basic four-polypeptidechain structure consisting of two heavy and two light chains, saidchains being stabilized, for example, by interchain disulfide bonds,which has the ability to specifically bind antigen. Both heavy and lightchains are folded into domains. The term “domain” refers to a globularregion of a heavy or light chain polypeptide comprising peptide loops(e.g., comprising 3 to 4 peptide loops) stabilized, for example, byβ-pleated sheet and/or interchain disulfide bond. Domains are furtherreferred to herein as “constant” or “variable”, based on the relativelack of sequence variation within the domains of various class membersin the case of a “constant” domain, or the significant variation withinthe domains of various class members in the case of a “variable” domain.“Constant” domains on the light chain are referred to interchangeably as“light chain constant regions”, “light chain constant domains”, “CL”regions or “CL” domains). “Constant” domains on the heavy chain arereferred to interchangeably as “heavy chain constant regions”, “heavychain constant domains”, “CH” regions or “CH” domains). “Variable”domains on the light chain are referred to interchangeably as “lightchain variable regions”, “light chain variable domains”, “VL” regions or“VL” domains). “Variable” domains on the heavy chain are referred tointerchangeably as “heavy chain constant regions”, “heavy chain constantdomains”, “CH” regions or “CH” domains).

The term “region” refers to a part or portion of an antibody chain andincludes constant or variable domains as defined herein, as well as morediscrete parts or portions of said domains. For example, light chainvariable domains or regions include “complementarity determiningregions” or “CDRs” interspersed among “framework regions” or “FRs”, asdefined herein.

Immunoglobulins or antibodies can exist in monomeric or polymeric form.The term “antigen-binding fragment” refers to a polypeptide fragment ofan immunoglobulin or antibody binds antigen or competes with intactantibody (i.e., with the intact antibody from which they were derived)for antigen binding (i.e., specific binding). The term “conformation”refers to the tertiary structure of a protein or polypeptide (e.g., anantibody, antibody chain, domain or region thereof). For example, thephrase “light (or heavy) chain conformation” refers to the tertiarystructure of a light (or heavy) chain variable region, and the phrase“antibody conformation” or “antibody fragment conformation” refers tothe tertiary structure of an antibody or fragment thereof.

“Specific binding” of an antibody mean that the antibody exhibitsappreciable affinity for antigen or a preferred epitope and, preferably,does not exhibit significant crossreactivity. “Appreciable” or preferredbinding include binding with an affinity of at least 10⁶, 10⁷, 10⁸, 10⁹M⁻¹, or 10¹⁰ M⁻¹. Affinities greater than 10⁷ M⁻¹, preferably greaterthan 10⁸ M⁻¹ are more preferred. Values intermediate of those set forthherein are also intended to be within the scope of the present inventionand a preferred binding affinity can be indicated as a range ofaffinities, for example, 10⁶ to 10¹³M⁻¹, preferably 10⁷ to 10¹³ M⁻¹,more preferably 10⁸ to 10¹³ M⁻¹. An antibody that “does not exhibitsignificant crossreactivity” is one that will not appreciably bind to anundesirable entity (e.g., an undesirable proteinaceous entity). Anantibody specific for a preferred epitope will, for example, notsignificantly crossreact with remote epitopes on the same protein orpeptide. Specific binding can be determined according to anyart-recognized means for determining such binding. Preferably, specificbinding is determined according to Scatchard analysis and/or competitivebinding assays.

In one embodiment, the invention provides for antibodies,antigen-binding fragments and/or antibody fragments of the inventionthat have high binding affinity for b-chemokines. High binding affinityrefers to binding affinities of, for example, about 10×10⁻¹²M (i.e. 10pM) or less. In one embodiment the immunoglobulins or antigen-bindingfragments have a Kd for binding to the b-chemokines (either binding aplurality of MCPs selected from MCP-1, MCP-2, and MCP-3 or that bind theindividual MCPs, i.e. an antibody or antigen-binding fragment that bindsto MCP-1, MCP-2 or MCP-3) between about 10×10⁻¹² M (10 pM) and about8×10⁻¹² M (8 pM) including 9×10⁻¹ M (9 pM); alternatively between about9×10⁻¹² M (9 pM) and about 7×10⁻¹² M (7 pM) including 8×10⁻¹² M (8 pM);alternatively between about 8×10⁻¹² M (8 pM) and about 6×10⁻¹² M (6 pM)including 7×10⁻¹² M (7 pM); alternatively between about 7×10⁻¹² M (7 pM)and about 5×10⁻¹² M (5 pM) including 6×10⁻¹² M (6 pM), alternativelybetween about 6×10⁻¹² M (6 pM) and about 4×10⁻¹² M (4 pM) including5×10⁻¹² M (5 pM), alternatively between about 5×10⁻¹² M (5 pM) and about3×10⁻¹² M (3 pM) including 4×10⁻¹² M (4 pM); alternatively between about4×10⁻¹² M (4 pM) and about 2×10⁻¹² M (2 pM) including 3×10⁻¹² M (3 pM);alternatively between about 3×10⁻¹² M (3 pM) and about 1×10⁻¹² M (1 pM)including 2×10⁻¹² M (2 pM); alternatively about 1×10⁻¹² M (1 pM) andabout 8×10⁻¹³ M (0.8 pM) including 9×10⁻¹³ M (0.9 pM); alternativelybetween about 9×10⁻¹³ M (0.9 pM) and about 7×10⁻¹³ M (0.7 pM) including8×10⁻¹³ M (0.8 pM); alternatively between about 8×10⁻¹³ M (0.8 pM) andabout 6×10⁻¹³ M (0.6 pM) including 7×10⁻¹³ M (0.7 pM); alternativelybetween about 7×10⁻¹³ M (0.7 pM) and about 5×10⁻¹³ M (0.5 pM) including6×10⁻¹³ M (0.6 pM), alternatively between about 6×10⁻¹³ M (0.6 pM) andabout 4×10⁻¹³ M (0.4 pM) including 5×10⁻¹³ M (0.5 pM), alternativelybetween about 5×10⁻¹³M (5 pM) and about 3×10⁻¹³ M (0.3 pM) including4×10⁻¹³ M (0.4 pM); alternatively between about 4×10⁻¹³ M (0.4 pM) andabout 2×10⁻¹³ M (0.2 pM) including 3×10⁻¹³ M (0.3 pM); alternativelybetween about 3×10⁻¹³M (0.3 pM) and about 1×10⁻¹³ M (0.1 pM) including2×10⁻¹³ M (0.2 pM). The invention would include for example an antibodyor antigen-binding fragment thereof that binds to MCP-1, MCP-2 or MCP-3wherein the antibody or antigen-binding fragment thereof has a Kd forbinding to MCP-1, MCP-2 or MCP-3 selected from the following Kd's: about10×10⁻¹³ M (1 pM), 9×10⁻¹³ M (0.9 pM), 8×10⁻¹³ M (0.8 pM), 7×10⁻¹³ M(0.7 pM), 6×10⁻¹³ M (0.6 pM), 5×10⁻¹³ M (0.5 pM), 4×10⁻¹³ M (0.4 pM),3×10⁻¹³ M (0.3 pM), 2×10⁻¹³ M (0.2 pM) or 1×10⁻¹³ M (0.1 pM). (Anexample of such an antibody would include for example 11K2 in which theantibody has a binding affinity for human MCP-1 of about 0.4 pM.) Theinvention would also include for example an antibody or antigen-bindingfragment thereof that binds to a plurality of MCP's (i.e. MCP-1 andMCP-2 or MCP-1 and MCP-3 or MCP-1, MCP-2 and MCP-3 or MCP-2 and MCP-3)wherein the antibody or antigen-binding fragment thereof has a Kd forbinding to at least one of the MCP's (i.e. MCP-1, MCP-2 or MCP-3)selected from the following Kd's: about 10×10⁻¹³ M (1 pM), 9×10⁻¹³ M(0.9 pM), 8×10⁻¹³ M (0.8 pM), 7×10⁻¹³ M (0.7 pM), 6×10⁻¹³ M (0.6 pM),5×10⁻¹³ M (0.5 pM), 4×10⁻¹³ M (0.4 pM), 3×10⁻¹³ M (0.3 pM), 2×10⁻¹³ M(0.2 pM) or 1×10⁻¹³ M (0.1 pM). (An example of such an antibody wouldalso include for example to 11K2 in which the antibody has a bindingaffinity for human MCP-1 of about 0.4 pM and also binds MCP-2 andMCP-3). Methods for measuring the binding affinity of the antibody,antigen-binding fragment and or antibody fragment for the variousb-chemokine(s) are known to those of skill in the art and include, forexample, the kinetic exclusion assay illustrated in the Examples aswell.

The invention also provides for immunoglobulins and antigen-bindingfragments comprising a Fab fragment wherein the Fab fragment has a Kdfor binding MCP-1, MCP-2 or MCP-3 of, for example, about 1.5×10⁻¹¹ M(i.e. 15 pM) or less. The invention would include for example anantibody, antigen-binding fragment and/or antibody fragment thereofwherein the Fab fragment has a Kd for binding to MCP-1, MCP-2 or MCP-3selected from the following Kd's: about 1.8×10⁻¹¹ M (18 pM), about1.7×10⁻¹¹ M (17 pM), about 1.6×10⁻¹¹ M (15 pM), about 1.5×10⁻¹¹ M (15pM), 1.4×10⁻¹¹ M (14 pM), 1.3×10⁻¹¹ M (13 pM), 1.2×10⁻¹¹ M (12 pM),1.1×10⁻¹¹ M (11 pM), 1×10⁻¹¹ M (10 pM), 0.9×10⁻¹¹ M (9 pM), 0.8×10⁻¹¹ M(8 pM), 0.7×10⁻¹¹ M (7 pM), 0.6×10⁻¹¹ M (6 pM), 0.5×10⁻¹¹ M (5 pM),0.4×10⁻¹¹ M (4 pM), 0.3×10⁻¹¹ M (3 pM), 0.2×10⁻¹¹ M (2 pM) or 0.1×10⁻¹¹M (1 pM). Methods for measuring the binding affinity of the antibody,antigen-binding fragment and or antibody fragment are known to those ofskill in the art and include, for example, the kinetic exclusion assayillustrated in Example 4 herewith.

The invention also provides for immunoglobulins and antigen-bindingfragments that have the following binding affinity for the b-chemokines(either binding a plurality of MCP's selected from MCP-1, MCP-2, andMCP-3 or that bind the individual MCPs, i.e. an antibody orantigen-binding fragment that binds to MCP-1, MCP-2 or MCP-3). In oneembodiment the binding affinity is between about 5×10⁻¹¹ M and about5×10⁻¹² M, in some embodiments the binding affinity is about 5×10⁻⁹ toabout 5×10⁻¹¹ M, in some embodiments the binding affinity is about5×10⁻⁷ M to about 5×10⁻⁸ M, in some embodiments the binding affinity isabout 5×10⁻⁸ M to about 5×10⁻⁹ M, in some embodiments the bindingaffinity is about 5×10⁻⁹ M to about 5×10⁻¹⁰ M, in some embodiments thebinding affinity is about 5×10⁻¹⁰ M to about 5×10⁻¹¹ M.

Binding fragments are produced by recombinant DNA techniques, or byenzymatic or chemical cleavage of intact immunoglobulins. Bindingfragments include Fab, Fab′, F(a′)₂, Fabc, Fv, single chains, andsingle-chain antibodies. Other than “bispecific” or “bifunctional”immunoglobulins or antibodies, an immunoglobulin or antibody isunderstood to have each of its binding sites identical. A “bispecific”or “bifunctional antibody” is an artificial hybrid antibody having twodifferent heavy/light chain pairs and two different binding sites.Bispecific antibodies can be produced by a variety of methods includingfusion of hybridomas or linking of Fab′ fragments. See, e.g.,Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelnyet al., J. Immunol. 148, 1547-1553 (1992).

The term “humanized immunoglobulin” or “humanized antibody” refers to animmunoglobulin or antibody that includes at least one humanizedimmunoglobulin or antibody chain (i.e., at least one humanized light orheavy chain). The term “humanized immunoglobulin chain” or “humanizedantibody chain” (i.e., a “humanized immunoglobulin light chain” or“humanized immunoglobulin heavy chain”) refers to an immunoglobulin orantibody chain (i.e., a light or heavy chain, respectively) having avariable region that includes a variable framework region substantiallyfrom a human immunoglobulin or antibody and complementarity determiningregions (CDRs) (e.g., at least one CDR, preferably two CDRs, morepreferably three CDRs) substantially from a non-human immunoglobulin orantibody, and further includes constant regions (e.g., at least oneconstant region or portion thereof, in the case of a light chain, andpreferably three constant regions in the case of a heavy chain). Theterm “humanized variable region” (e.g., “humanized light chain variableregion” or “humanized heavy chain variable region”) refers to a variableregion that includes a variable framework region substantially from ahuman immunoglobulin or antibody and complementarity determining regions(CDRs) substantially from a non-human immunoglobulin or antibody.

The phrase “substantially from a human immunoglobulin or antibody” or“substantially human” means that, when aligned to a human immunoglobulinor antibody amino sequence for comparison purposes, the region shares atleast 80-90%, preferably 90-95%, more preferably 95-99% identity (i.e.,local sequence identity) with the human framework or constant regionsequence, allowing, for example, for conservative substitutions,consensus sequence substitutions, germline substitutions, backmutations,and the like. The introduction of conservative substitutions, consensussequence substitutions, germline substitutions, backmutations, and thelike, is often referred to as “optimization” of a humanized antibody orchain. The phrase “substantially from a non-human immunoglobulin orantibody” or “substantially non-human” means having an immunoglobulin orantibody sequence at least 80-95%, preferably 90-95%, more preferably,96%, 97%, 98%, or 99% identical to that of a non-human organism, e.g., anon-human mammal.

Accordingly, all regions or residues of a humanized immunoglobulin orantibody, or of a humanized immunoglobulin or antibody chain, exceptpossibly the CDRs, are substantially identical to the correspondingregions or residues of one or more native human immunoglobulinsequences. The term “corresponding region” or “corresponding residue”refers to a region or residue on a second amino acid or nucleotidesequence which occupies the same (i.e., equivalent) position as a regionor residue on a first amino acid or nucleotide sequence, when the firstand second sequences are optimally aligned for comparison purposes.

The terms “humanized immunoglobulin” or “humanized antibody” are notintended to encompass chimeric immunoglobulins or antibodies, as definedinfra. Although humanized immunoglobulins or antibodies are chimeric intheir construction (i.e., comprise regions from more than one species ofprotein), they include additional features (i.e., variable regionscomprising donor CDR residues and acceptor framework residues) not foundin chimeric immunoglobulins or antibodies, as defined herein.

The term “significant identity” means that two polypeptide sequences,when optimally aligned, such as by the programs GAP or BESTFIT usingdefault gap weights, share at least 50-60% sequence identity, preferably60-70% sequence identity, more preferably 70-80% sequence identity, morepreferably at least 80-90% identity, even more preferably at least90-95% identity, and even more preferably at least 95% sequence identityor more (e.g., 99% sequence identity or more). The term “substantialidentity” means that two polypeptide sequences, when optimally aligned,such as by the programs GAP or BESTFIT using default gap weights, shareat least 80-90% sequence identity, preferably 90-95% sequence identity,and more preferably at least 95% sequence identity or more (e.g., 99%sequence identity or more). For sequence comparison, typically onesequence acts as a reference sequence, to which test sequences arecompared. When using a sequence comparison algorithm, test and referencesequences are input into a computer, subsequence coordinates aredesignated, if necessary, and sequence algorithm program parameters aredesignated. The sequence comparison algorithm then calculates thepercent sequence identity for the test sequence(s) relative to thereference sequence, based on the designated program parameters. Theterms “sequence identity” and “sequence identity” are usedinterchangeably herein.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generallyAusubel et al., Current Protocols in Molecular Biology). One example ofalgorithm that is suitable for determining percent sequence identity andsequence similarity is the BLAST algorithm, which is described inAltschul et al., J. Mol. Biol. 215:403 (1990). Software for performingBLAST analyses is publicly available through the National Center forBiotechnology Information (publicly accessible through the NationalInstitutes of Health NCBI internet server). Typically, default programparameters can be used to perform the sequence comparison, althoughcustomized parameters can also be used. For amino acid sequences, theBLASTP program uses as defaults a wordlength (W) of 3, an expectation(E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff,Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

Preferably, residue positions which are not identical differ byconservative amino acid substitutions. For purposes of classifying aminoacids substitutions as conservative or nonconservative, amino acids aregrouped as follows: Group I (hydrophobic sidechains): leu, met, ala,val, leu, ile; Group II (neutral hydrophilic side chains): cys, ser,thr; Group III (acidic side chains): asp, glu; Group IV (basic sidechains): asn, gin, his, lys, arg; Group V (residues influencing chainorientation): gly, pro; and Group VI (aromatic side chains): trp, tyr,phe. Conservative substitutions involve substitutions between aminoacids in the same class. Non-conservative substitutions constituteexchanging a member of one of these classes for a member of another.

Preferably, humanized immunoglobulins or antibodies bind antigen with anaffinity that is within a factor of three, four, or five of that of thecorresponding non-human antibody. For example, if the nonhuman antibodyhas a binding affinity of 10⁹ M⁻¹, humanized antibodies will have abinding affinity of at least 3×10⁹ M⁻¹, 4×10⁹ M⁻¹ or 10⁹ M⁻¹. Whendescribing the binding properties of an immunoglobulin or antibodychain, the chain can be described based on its ability to “directantigen (e.g, MCP-1) binding”. A chain is said to “direct antigenbinding” when it confers upon an intact immunoglobulin or antibody (orantigen binding fragment thereof) a specific binding property or bindingaffinity. A mutation (e.g., a backmutation) is said to substantiallyaffect the ability of a heavy or light chain to direct antigen bindingif it affects (e.g., decreases) the binding affinity of an intactimmunoglobulin or antibody (or antigen binding fragment thereof)comprising said chain by at least an order of magnitude compared to thatof the antibody (or antigen binding fragment thereof) comprising anequivalent chain lacking said mutation. A mutation “does notsubstantially affect (e.g., decrease) the ability of a chain to directantigen binding” if it affects (e.g., decreases) the binding affinity ofan intact immunoglobulin or antibody (or antigen binding fragmentthereof) comprising said chain by only a factor of two, three, or fourof that of the antibody (or antigen binding fragment thereof) comprisingan equivalent chain lacking said mutation.

The term “chimeric immunoglobulin” or antibody refers to animmunoglobulin or antibody whose light and heavy chains are derived fromdifferent species. Chimeric immunoglobulins or antibodies can beconstructed, for example by genetic engineering, from immunoglobulingene segments belonging to different species.

An “antigen” is an entity (e.g., a proteinaceous entity or peptide) towhich an antibody specifically binds.

The term “epitope” or “antigenic determinant” refers to a site on anantigen to which an immunoglobulin or antibody (or antigen bindingfragment thereof) specifically binds. Epitopes can be formed both fromcontiguous amino acids or noncontiguous amino acids juxtaposed bytertiary folding of a protein. Epitopes formed from contiguous aminoacids are typically retained on exposure to denaturing solvents whereasepitopes formed by tertiary folding are typically lost on treatment withdenaturing solvents. An epitope typically includes at least 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in a unique spatialconformation. Methods of determining spatial conformation of epitopesinclude, for example, x-ray crystallography and 2-dimensional nuclearmagnetic resonance. See, e.g., Epitope Mapping Protocols in Methods inMolecular Biology, Vol. 66, G. E. Morris, Ed. (1996).

Antibodies that recognize the same epitope can be identified in a simpleimmunoassay showing the ability of one antibody to block the binding ofanother antibody to a target antigen, i.e., a competitive binding assay.Competitive binding is determined in an assay in which theimmunoglobulin under test inhibits specific binding of a referenceantibody to a common antigen, such as MCP-1. Numerous types ofcompetitive binding assays are known, for example: solid phase direct orindirect radioimmunoassay (RIA), solid phase direct or indirect enzymeimmunoassay (EIA), sandwich competition assay (see Stahil et al.,Methods in Enzymology 9:242 (1983)); solid phase direct biotin-avidinETA (see Kirkland et al., J. Immunol. 137:3614 (1986)); solid phasedirect labeled assay, solid phase direct labeled sandwich assay (seeHarlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborPress (1988)); solid phase direct label RIA using I-125 label (see Morelet al., Mol. Immunol. 2.5(1):7 (1988)); solid phase direct biotin-avidinETA (Cheung et al., Virology 176:546 (1990)); and direct labeled RIA.(Moldenhauer et al., Scand. J. Immunol. 32:77 (1990)). Typically, suchan assay involves the use of purified antigen bound to a solid surfaceor cells bearing either of these, an unlabeled test immunoglobulin and alabeled reference immunoglobulin. Competitive inhibition is measured bydetermining the amount of label bound to the solid surface or cells inthe presence of the test immunoglobulin. Usually the test immunoglobulinis present in excess. Usually, when a competing antibody is present inexcess, it will inhibit specific binding of a reference antibody to acommon antigen by at least 50-55%, 55-60%, 60-65%, 65-70% 70-75% ormore.

An epitope is also recognized by immunologic cells, for example, B cellsand/or T cells. Cellular recognition of an epitope can be determined byin vitro assays that measure antigen-dependent proliferation, asdetermined by ³H-thymidine incorporation, by cytokine secretion, byantibody secretion, or by antigen-dependent killing (cytotoxic Tlymphocyte assay).

Exemplary epitopes or antigenic determinants can be found within humanMCP molecules, and are preferably within MCP-1, MCP-2, and MCP-3. Otherpreferred epitopes are those that are commonly found within MCP-1 andMCP-2, MCP-1 and MCP-3, MCP-1 and MCP-3, and MCP-1, MCP-2 and MCP-3.

As used herein, the term “b-chemokine” refers to a polypeptidecontaining four conserved cysteine residues characteristic ofb-chemokines (e.g., as described in inflammation (Van Coillie et al.(1999) Cytokine & Growth Factor Rev. 10:61-86) wherein the first twoconserved cysteines are adjacent.

As used herein, the term “inhibiting the activity of b-chemokines”refers to causing a decrease in the relative activity of b-chemokines inthe presence of the antibody or antigen-binding fragment thereof incomparison with the activity observed in the absence of the antibody orantigen-binding fragment thereof. The term “inhibits MCP activity” isdefined herein as reducing or eliminating activity associated with MCPs,e.g. MCP-1, MCP-2, or MCP-3, for example by reducing or inhibitingMCP-induced chemotaxis and/or by reducing or inhibiting MCP-inducedcollagen expression and/or by reducing or inhibiting MCP-inducedangiogenesis. Activities associated with MCP-induction can be assayedaccording to standard methods known in the art, and as described herein.

As used herein, the term “sign of an inflammatory disorder” refers toobservable or measurable indications of pathological inflammation,including, but not limited to edema, fever, emigration of leukocytes,proliferation of blood vessels, proliferation of connective tissue,redness, localized heat, exudation, and other signs as described inROBBINS PATHOLOGIC BASIS OF DISEASE, 4^(TH) EDITION, R. S. Cotran etal., Eds. W.B. Saunders, Co., 1989.

As used herein, the term “blocking chemotaxis” refers to a decrease inthe relative amount of chemotactic activity of cells in the presence ofthe antibody or antigen-binding fragment thereof in comparison withchemotactic activity observed in the absence of the antibody orantigen-binding fragment thereof.

As used herein, the term “MCP MRHAS Motif” refers to an amino acid motifin human MCP-1 and MCP-3 termed Meningitis Related Homologous AntigenicSequence. For human MCP-1, the MRHAS amino acid motif isGln-Thr-Gln-Thr-Pro-Lys-Thr (SEQ ID NO:1); and for human MCP-3, theMRHAS motif is Lys-Thr-Gln-Thr-Pro-Lys-Leu (SEQ ID NO:2).

The term “effective dose” or “effective dosage” is defined as an amountsufficient to achieve or at least partially achieve the desired effect.The term “therapeutically effective dose” is defined as an amountsufficient to cure or at least partially arrest the disease and itscomplications in a subject already suffering from the disease. Amountseffective for this use will depend upon the severity of the infectionand the general state of the subject's own immune system.

The term “subject” includes human and other mammalian subjects thatreceive either prophylactic or therapeutic treatment.

I. Immunological and Therapeutic Reagents

Immunological and therapeutic reagents of the invention comprise orconsist of immunogens or antibodies, or functional or antigen bindingfragments thereof, as defined herein. The basic antibody structural unitis known to comprise a tetramer of subunits. Each tetramer is composedof two identical pairs of polypeptide chains, each pair having one“light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa). Theamino-terminal portion of each chain includes a variable region of about100 to 110 or more amino acids primarily responsible for antigenrecognition. The carboxy-terminal portion of each chain defines aconstant region primarily responsible for effector function.

Light chains are classified as either kappa or lambda and are about 230residues in length. Heavy chains are classified as gamma (γ), mu (μ),alpha (α), delta (δ), or epsilon (ε), are about 450-600 residues inlength, and define the antibody's isotype as IgG, IgM, IgA, IgD and IgE,respectively. Both heavy and light chains are folded into domains. Theterm “domain” refers to a globular region of a protein, for example, animmunoglobulin or antibody. Immunoglobulin or antibody domains include,for example, 3 or four peptide loops stabilized by β-pleated sheet andan interchain disulfide bond. Intact light chains have, for example, twodomains (V_(L) and C_(L)) and intact heavy chains have, for example,four or five domains (V_(H), C_(H)1, C_(H)2, and C_(H)3).

Within light and heavy chains, the variable and constant regions arejoined by a “J” region of about 12 or more amino acids, with the heavychain also including a “D” region of about 10 more amino acids. (Seegenerally, Fundamental Immunology (Paul, W., ed., 2nd ed. Raven Press,N.Y. (1989), Ch. 7, incorporated by reference in its entirety for allpurposes).

The variable regions of each light/heavy chain pair form the antibodybinding site. Thus, an intact antibody has two binding sites. Except inbifunctional or bispecific antibodies, the two binding sites are thesame. The chains all exhibit the same general structure of relativelyconserved framework regions (FR) joined by three hypervariable regions,also called complementarity determining regions or CDRs.Naturally-occurring chains or recombinantly produced chains can beexpressed with a leader sequence which is removed during cellularprocessing to produce a mature chain. Mature chains can also berecombinantly produced having a non-naturally occurring leader sequence,for example, to enhance secretion or alter the processing of aparticular chain of interest.

The CDRs of the two mature chains of each pair are aligned by theframework regions, enabling binding to a specific epitope. FromN-terminal to C-terminal, both light and heavy chains comprise thedomains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. “FR4” also is referredto in the art as the D/J region of the variable heavy chain and the Jregion of the variable light chain. The assignment of amino acids toeach domain is in accordance with the definitions of Kabat, Sequences ofProteins of Immunological Interest (National Institutes of Health,Bethesda, Md., 1987 and 1991). An alternative structural definition hasbeen proposed by Chothia et al., J. Mol. Biol. 196:901 (1987); Nature342:878 (1989); and J. Mol. Biol. 186:651 (1989) (hereinaftercollectively referred to as “Chothia et al.” and incorporated byreference in their entirety for all purposes).

A. MCP Antibodies

Therapeutic agents of the invention include antibodies that specificallybind to MCPs or other b-chemokines. Such antibodies can be monoclonal orpolyclonal. Some such antibodies bind specifically MCP-1. Some bindspecifically to MCP-2. Some bind to both MCP-1 and MCP-2. Some suchantibodies bind to MCP-3. Antibodies used in therapeutic methodspreferably have an intact constant region or at least sufficient of theconstant region to interact with an Fc receptor. Human isotype IgG1 ispreferred because of it having highest affinity of human isotypes forthe FcRI receptor on phagocytic cells. Bispecific Fab fragments can alsobe used, in which one arm of the antibody has specificity for MCP-1,MCP-2, MCP-3, or a combination thereof, and the other for an Fcreceptor. Preferred antibodies bind to MCP-1 with a binding affinitygreater than (or equal to) about 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹²,or 10¹³ M⁻¹ (including affinities intermediate of these values).

In certain embodiments the antibodies of the invention, and fragmentsthereof, bind to regions in the b-chemokines (e.g., MCP-1, MCP-2 andMCP-3). In some embodiments, the antibodies or antigen-binding fragmentsthereof bind MCP-2 and at least one other b-chemokine (e.g., MCP-1 orMCP-3). Thus in some embodiments, the antibodies or fragments thereofbind MCP-1 and MCP-2 and in other embodiments the antibodies orfragments thereof bind MCP2 and MCP-3. In other embodiments, theantibodies or antigen-binding fragments thereof bind MCP-1, MCP-2 andMCP-3. In other embodiments, the antibodies or antigen-binding fragmentsthereof bind MCP-1 and MCP-3 other than to regions containing the MRHASmotifs, QTQTPKT (MCP-1) and KTQTPKL (MCP-3).

In certain embodiments, the antibodies or antigen-binding fragmentsthereof comprise antibodies selected from the group consisting of 11K2.1(ATCC Accession No. PTA-3987), 6D21.1 (ATCC Accession No. PTA-3989),4N4.1 (ATCC Accession No. PTA-3994), 5A13.1 (ATCC Accession No.PTA-3995), 7H11.1 (ATCC Accession No. PTA-3985), 1A1.1 (ATCC AccessionNo. PTA-3990), 6I5.1 (ATCC Accession No. PTA-3986), 2O24.1 (ATCCAccession No. PTA-3993), 9B11.1 (ATCC Accession No. PTA-3992), 9B12.1(ATCC Accession No. PTA-3996), 9C11.1 (ATCC Accession No. PTA-3988), and12F15.1 (ATCC Accession No. PTA-3991), or antigen-binding fragments ofthese antibodies.

Polyclonal sera typically contain mixed populations of antibodiesbinding to several epitopes, including, for example, MCP-1. Monoclonalantibodies bind to a specific epitope within b-chemokines, e.g., MCP-1,that can be a conformational or nonconformational epitope. Preferredmonoclonal antibodies bind to an epitope within MCP-1 or MCP-2. Somepreferred monoclonal antibodies bind to an epitope within MCP-1 andMCP-2, and some to an epitope within MCP-1, MCP-2, and MCP-3. It isrecommended that such antibodies be screened for activity in the mousemodels before use.

When an antibody is said to bind to an epitope within specified residuesof a certain b-chemokine, such as MCP-1 for example, what is meant isthat the antibody specifically binds to a polypeptide containing thespecified residues. Such an antibody does not necessarily contact everyresidue, nor does every single amino acid substitution or deletion withthe specified necessarily significantly affect binding affinity. Epitopespecificity of an antibody can be determined, for example, by forming aphage display library in which different members display differentsubsequences of MCPs. The phage display library is then selected formembers specifically binding to an antibody under test. A family ofsequences is isolated. Typically, such a family contains a common coresequence, and varying lengths of flanking sequences in differentmembers. The shortest core sequence showing specific binding to theantibody defines the epitope bound by the antibody. Antibodies can alsobe tested for epitope specificity in a competition assay with anantibody whose epitope specificity has already been determined. Forexample, antibodies that compete with the 11K2 antibody for binding toMCP-1 bind to the same or similar epitope as 11K2. Likewise antibodiesthat compete with the 1A1 antibody bind to the same or similar epitope.Screening antibodies for epitope specificity is a useful predictor oftherapeutic efficacy. In one embodiment, the invention includes ananti-MCP antibody with a heavy chain which contacts residues R30, T32,S34, K38, E39, V41, P55, K56, Q61, M64 of MCP-1. The invention alsoincludes a an anti-MCP antibody with a light chain which contactsresidues D65, D68, K69 of MCP-1.

1. Production of Nonhuman Antibodies

The present invention features non-human antibodies, for example,antibodies having specificity for the preferred MCP epitopes of theinvention. Such antibodies can be used in formulating varioustherapeutic compositions of the invention or, preferably, providecomplementarity determining regions for the production of humanized orchimeric antibodies (described in detail below). The production ofnon-human monoclonal antibodies, e.g., murine, guinea pig, primate,rabbit or rat, can be accomplished by, for example, immunizing theanimal with at least one b-chemokine. In producing the antibodies of theinvention, the immunogens may be a preparation containing at least oneb-chemokine, preferably more than one b-chemokine. In some embodiments,the b-chemokines are human monocyte chemotactic proteins (MCPs),including MCP-1, MCP-2 and MCP-3. The b-chemokines may be native orrecombinantly produced b-chemokines. In some embodiments, the immunogensmay be antigenic fragments of b-chemokines, such as fragments of MCPswhich may optionally be conjugated to a carrier molecule to impart astronger immune response upon administration to an animal. Theb-chemokine immunogens, such as MCPs may contain additions, deletionsand/or substitutions of amino acids, provided that the alterations donot ablate antigenicity of the mutated b-chemokines such that antibodiesagainst the mutant versions do not bind native b-chemokines. Preferably,the amino acid substitutions are conservative changes in the amino acidsequence, provided the MCP molecules remain antigenic. See Harlow &Lane, supra, incorporated by reference for all purposes).

Such an immunogen can be obtained from a natural source, by peptidesynthesis or by recombinant expression. Optionally, the immunogen can beadministered fused or otherwise complexed with a carrier protein, asdescribed below. Optionally, the immunogen can be administered with anadjuvant. The term “adjuvant” refers to a compound that whenadministered in conjunction with an antigen augments the immune responseto the antigen, but when administered alone does not generate an immuneresponse to the antigen. Adjuvants can augment an immune response byseveral mechanisms including lymphocyte recruitment, stimulation of Band/or T cells, and stimulation of macrophages. Several types ofadjuvant can be used as described below. Complete Freund's adjuvantfollowed by incomplete adjuvant is preferred for immunization oflaboratory animals.

Rabbits or guinea pigs are typically used for making polyclonalantibodies. Exemplary preparation of polyclonal antibodies, e.g., forpassive protection, can be performed as follows. 125 non-transgenic miceare immunized with 100 μg of a MCP-1/MCP-2 cocktail, plus CFA/IFAadjuvant, and euthanized at 4-5 months. Blood is collected fromimmunized mice. IgG is separated from other blood components. Antibodyspecific for the immunogen may be partially purified by affinitychromatography. An average of about 0.5-1 mg of immunogen-specificantibody is obtained per mouse, giving a total of 60-120 mg.

Mice are typically used for making monoclonal antibodies. Monoclonalscan be prepared against a fragment by injecting, for example, a fragmentof MCP-1 into a mouse, preparing hybridomas and screening the hybridomasfor an antibody that specifically binds to MCP-1. Optionally, antibodiesare screened for binding to a specific region or desired fragment ofMCP-1 without binding to other nonoverlapping fragments of MCP-1. Thelatter screening can be accomplished by determining binding of anantibody to a collection of deletion mutants of a MCP-1 peptide anddetermining which deletion mutants bind to the antibody. Binding can beassessed, for example, by Western blot or ELISA. The smallest fragmentto show specific binding to the antibody defines the epitope of theantibody. Alternatively, epitope specificity can be determined by acompetition assay is which a test and reference antibody compete forbinding to MCP-1. If the test and reference antibodies compete, thenthey bind to the same epitope or epitopes sufficiently proximal suchthat binding of one antibody interferes with binding of the other. Thepreferred isotype for such antibodies is mouse isotype IgG2a orequivalent isotype in other species. Mouse isotype IgG2a is theequivalent of human isotype IgG1.

2. Chimeric and Humanized Antibodies

The present invention also features chimeric and/or humanized antibodies(i.e., chimeric and/or humanized immunoglobulins) specific forb-chemokines, including MCPs. Chimeric and/or humanized antibodies havethe same or similar binding specificity and affinity as a mouse or othernonhuman antibody that provides the starting material for constructionof a chimeric or humanized antibody.

In one embodiment, the CDRs of the 11K2 antibody can be used to producehumanized and chimeric antibodies. The invention features an isolatedantibody, or antigen binding portion thereof, which binds a plurality ofb-chemokines, wherein said b-chemokines comprise MCP-2 and at least oneother b-chemokine, which comprises at least one of the following CDRs:CDR1, CDR2, or CDR3, from the 11K2 heavy chain variable region describedin SEQ ID NO: 27. In an additional embodiment, the invention features anisolated antibody, or antigen binding portion thereof which binds aplurality of b-chemokines, wherein said b-chemokines comprise MCP-2 andat least one other b-chemokine, which comprises at least one of thefollowing CDR combinations: CDR1 and CDR2; CDR1 and CDR3; CDR2 and CDR3;or CDR1, CDR2, and CDR3, from the 11K2 heavy chain variable regiondescribed in SEQ ID NO: 27. In one embodiment, the antibody of theinvention is a chimeric antibody. In another embodiment, the antibody ofthe invention is a humanized antibody.

The CDRs of the 1A1 antibody can be also used to produce humanized andchimeric antibodies. In one embodiment, the invention provides anisolated antibody, or antigen binding portion thereof, which binds aplurality of b-chemokines, wherein said b-chemokines comprise MCP-2 andat least one other b-chemokine, which comprises at least one of thefollowing CDRs: CDR1, CDR2, or CDR3, from the 1A1 heavy chain variableregion described in SEQ ID NO: 11. In another embodiment, the inventionprovides an isolated antibody, or antigen binding portion thereof, whichbinds a plurality of b-chemokines, wherein said b-chemokines compriseMCP-2 and at least one other b-chemokine, which comprises at least oneof the following CDR combinations: CDR1 and CDR2; CDR1 and CDR3; CDR2and CDR3; and CDR1, CDR2, and CDR3, from the 1A1 heavy chain variableregion described in SEQ ID NO: 11. In one embodiment, the antibody ofthe invention is a chimeric antibody. In another embodiment, theantibody of the invention is a humanized antibody.

a. Production of Chimeric Antibodies.

The term “chimeric antibody” refers to an antibody whose light and heavychain genes have been constructed, typically by genetic engineering,from immunoglobulin gene segments belonging to different species. Forexample, the variable (V) segments of the genes from a mouse monoclonalantibody may be joined to human constant (C) segments, such as IgG1 andIgG4. Human isotype IgG1 is preferred. A typical chimeric antibody isthus a hybrid protein consisting of the V or antigen-binding domain froma mouse antibody and the C or effector domain from a human antibody.

b. Production of Humanized Antibodies

The term “humanized antibody” refers to an antibody comprising at leastone chain comprising variable region framework residues substantiallyfrom a human antibody chain (referred to as the acceptor immunoglobulinor antibody) and at least one complementarity determining regionsubstantially from a mouse-antibody, (referred to as the donorimmunoglobulin or antibody). See, Queen et al., Proc. Natl. Acad. Sci.USA 86:10029-10033 (1989), U.S. Pat. No. 5,530,101, U.S. Pat. No.5,585,089, U.S. Pat. No. 5,693,761, U.S. Pat. No. 5,693,762, Selick etal., WO 90/07861, and Winter, U.S. Pat. No. 5,225,539 (incorporated byreference in their entirety for all purposes). The constant region(s),if present, are also substantially or entirely from a humanimmunoglobulin.

The substitution of mouse CDRs into a human variable domain framework ismost likely to result in retention of their correct spatial orientationif the human variable domain framework adopts the same or similarconformation to the mouse variable framework from which the CDRsoriginated. This is achieved by obtaining the human variable domainsfrom human antibodies whose framework sequences exhibit a high degree ofsequence identity with the murine variable framework domains from whichthe CDRs were derived. The heavy and light chain variable frameworkregions can be derived from the same or different human antibodysequences. The human antibody sequences can be the sequences ofnaturally occurring human antibodies or can be consensus sequences ofseveral human antibodies. See Kettleborough et al., Protein Engineering4:773 (1991); Kolbinger et al., Protein Engineering 6:971 (1993) andCarter et al., WO 92/22653.

Having identified the complementarity determining regions of the murinedonor immunoglobulin and appropriate human acceptor immunoglobulins, thenext step is to determine which, if any, residues from these componentsshould be substituted to optimize the properties of the resultinghumanized antibody. In general, substitution of human amino acidresidues with marine should be minimized, because introduction of murineresidues increases the risk of the antibody eliciting ahuman-anti-mouse-antibody (HAMA) response in humans. Art-recognizedmethods of determining immune response can be performed to monitor aHAMA response in a particular subject or during clinical trials.Subjects administered humanized antibodies can be given animmunogenicity assessment at the beginning and throughout theadministration of said therapy. The HAMA response is measured, forexample, by detecting antibodies to the humanized therapeutic reagent,in serum samples from the subject using a method known to one in theart, including surface plasmon resonance technology (BIACORE) and/orsolid-phase ELISA analysis.

Certain amino acids from the human variable region framework residuesare selected for substitution based on their possible influence on CDRconformation and/or binding to antigen. The unnatural juxtaposition ofmurine CDR regions with human variable framework region can result inunnatural conformational restraints, which, unless corrected bysubstitution of certain amino acid residues, lead to loss of bindingaffinity.

The selection of amino acid residues for substitution is determined, inpart, by computer modeling. Computer hardware and software are describedherein for producing three-dimensional images of immunoglobulinmolecules. In general, molecular models are produced starting fromsolved structures for immunoglobulin chains or domains thereof. Thechains to be modeled are compared for amino acid sequence similaritywith chains or domains of solved three-dimensional structures, and thechains or domains showing the greatest sequence similarity is/areselected as starting points for construction of the molecular model.Chains or domains sharing at least 50% sequence identity are selectedfor modeling, and preferably those sharing at least 60%, 70%, 80%, 90%sequence identity or more are selected for modeling. The solved startingstructures are modified to allow for differences between the actualamino acids in the immunoglobulin chains or domains being modeled, andthose in the starting structure. The modified structures are thenassembled into a composite immunoglobulin. Finally, the model is refinedby energy minimization and by verifying that all atoms are withinappropriate distances from one another and that bond lengths and anglesare within chemically acceptable limits.

The selection of amino acid residues for substitution can also bedetermined, in part, by examination of the characteristics of the aminoacids at particular locations, or empirical observation of the effectsof substitution or mutagenesis of particular amino acids. For example,when an amino acid differs between a murine variable region frameworkresidue and a selected human variable region framework residue, thehuman framework amino acid should usually be substituted by theequivalent framework amino acid from the mouse antibody when it isreasonably expected that the amino acid:

-   -   (1) noncovalently binds antigen directly,    -   (2) is adjacent to a CDR region,    -   (3) otherwise interacts with a CDR region (e.g., is within about        3-6 Å of a CDR region as determined by computer modeling), or    -   (4) participates in the VL-VH interface.

Residues which “noncovalently bind antigen directly” include amino acidsin positions in framework regions which are have a good probability ofdirectly interacting with amino acids on the antigen according toestablished chemical forces, for example, by hydrogen bonding, Van derWaals forces, hydrophobic interactions, and the like.

CDR and framework regions are as defined by Kabat et al. or Chothia etal., supra. When framework residues, as defined by Kabat et al., supra,constitute structural loop residues as defined by Chothia et al., supra,the amino acids present in the mouse antibody may be selected forsubstitution into the humanized antibody. Residues which are “adjacentto a CDR region” include amino acid residues in positions immediatelyadjacent to one or more of the CDRs in the primary sequence of thehumanized immunoglobulin chain, for example, in positions immediatelyadjacent to a CDR as defined by Kabat, or a CDR as defined by Chothia(See e.g., Chothia and Lesk J M B 196:901 (1987)). These amino acids areparticularly likely to interact with the amino acids in the CDRs and, ifchosen from the acceptor, to distort the donor CDRs and reduce affinity.Moreover, the adjacent amino acids may interact directly with theantigen (Amit et al., Science, 233:747 (1986), which is incorporatedherein by reference) and selecting these amino acids from the donor maybe desirable to keep all the antigen contacts that provide affinity inthe original antibody.

Residues that “otherwise interact with a CDR region” include those thatare determined by secondary structural analysis to be in a spatialorientation sufficient to effect a CDR region. In one embodiment,residues that “otherwise interact with a CDR region” are identified byanalyzing a three-dimensional model of the donor immunoglobulin (e.g., acomputer-generated model). A three-dimensional model, typically of theoriginal donor antibody, shows that certain amino acids outside of theCDRs are close to the CDRs and have a good probability of interactingwith amino acids in the CDRs by hydrogen bonding, Van der Waals forces,hydrophobic interactions, etc. At those amino acid positions, the donorimmunoglobulin amino acid rather than the acceptor immunoglobulin aminoacid may be selected. Amino acids according to this criterion willgenerally have a side chain atom within about 3 angstrom units (A) ofsome atom in the CDRs and must contain an atom that could interact withthe CDR atoms according to established chemical forces, such as thoselisted above.

In the case of atoms that may form a hydrogen bond, the 3 Å is measuredbetween their nuclei, but for atoms that do not form a bond, the 3 Å ismeasured between their Van der Waals surfaces. Hence, in the lattercase, the nuclei must be within about 6 Å (3 Å plus the sum of the Vander Waals radii) for the atoms to be considered capable of interacting.In many cases the nuclei will be from 4 or 5 to 6 Å apart. Indetermining whether an amino acid can interact with the CDRs, it ispreferred not to consider the last 8 amino acids of heavy chain CDR 2 aspart of the CDRs, because from the viewpoint of structure, these 8 aminoacids behave more as part of the framework.

Amino acids that are capable of interacting with amino acids in theCDRs, may be identified in yet another way. The solvent accessiblesurface area of each framework amino acid is calculated in two ways: (1)in the intact antibody, and (2) in a hypothetical molecule consisting ofthe antibody with its CDRs removed. A significant difference betweenthese numbers of about 10 square angstroms or more shows that access ofthe framework amino acid to solvent is at least partly blocked by theCDRs, and therefore that the amino acid is making contact with the CDRs.Solvent accessible surface area of an amino acid may be calculated basedon a three-dimensional model of an antibody, using algorithms known inthe art (e.g., Connolly, J. Appl. Cryst. 16:548 (1983) and Lee andRichards, J. Mol. Biol. 55:379 (1971), both of which are incorporatedherein by reference). Framework amino acids may also occasionallyinteract with the CDRs indirectly, by affecting the conformation ofanother framework amino acid that in turn contacts the CDRs.

The amino acids at several positions in the framework are known to becapable of interacting with the CDRs in many antibodies (Chothia andLesk, supra, Chothia et al., supra and Tramontano et al., J. Mol. Biol.215:175 (1990), all of which are incorporated herein by reference).Notably, the amino acids at positions 2, 48, 64 and 71 of the lightchain and 26-30, 71 and 94 of the heavy chain (numbering according toKabat) are known to be capable of interacting with the CDRs in manyantibodies. The amino acids at positions 35 in the light chain and 93and 103 in the heavy chain are also likely to interact with the CDRs. Atall these numbered positions, choice of the donor amino acid rather thanthe acceptor amino acid (when they differ) to be in the humanizedimmunoglobulin is preferred. On the other hand, certain residues capableof interacting with the CDR region, such as the first 5 amino acids ofthe light chain, may sometimes be chosen from the acceptorimmunoglobulin without loss of affinity in the humanized immunoglobulin.

Residues which “participate in the VL-VH interface” or “packingresidues” include those residues at the interface between VL and VH asdefined, for example, by Novotny and Haber, Proc. Natl. Acad. Sci. USA,82:4592-66 (1985) or Chothia et al, supra. Generally, unusual packingresidues should be retained in the humanized antibody if they differfrom those in the human frameworks.

In general, one or more of the amino acids fulfilling the above criteriais substituted. In some embodiments, all or most of the amino acidsflailing the above to criteria are substituted. Occasionally, there issome ambiguity about whether a particular amino acid meets the abovecriteria, and alternative variant immunoglobulins are produced, one ofwhich has that particular substitution, the other of which does not.Alternative variant immunoglobulins so produced can be tested in any ofthe assays described herein for the desired activity, and the preferredimmunoglobulin selected.

Usually the CDR regions in humanized antibodies are substantiallyidentical, and more usually, identical to the corresponding CDR regionsof the donor antibody. Although not usually desirable, it is sometimespossible to make one or more conservative amino acid substitutions ofCDR residues without appreciably affecting the binding affinity of theresulting humanized immunoglobulin. By conservative substitutions isintended combinations such as gly, ala; val, ile, leu; asp, glu; asn,gln; ser, thr; lys, arg; and phe, tyr.

Additional candidates for substitution are acceptor human frameworkamino acids that are “unusual” or “rare” for a human immunoglobulin atthat position. These amino acids can be substituted with amino acidsfrom the equivalent position of the mouse donor antibody or from theequivalent positions of more typical human immunoglobulins. For example,substitution may be desirable when the amino acid in a human frameworkregion of the acceptor immunoglobulin is rare for that position and thecorresponding amino acid in the donor immunoglobulin is common for thatposition in human immunoglobulin sequences; or when the amino acid inthe acceptor immunoglobulin is rare for that position and thecorresponding amino acid in the donor immunoglobulin is also rare,relative to other human sequences. These criterion help ensure that anatypical amino acid in the human framework does not disrupt the antibodystructure. Moreover, by replacing an unusual human acceptor amino acidwith an amino acid from the donor antibody that happens to be typicalfor human antibodies, the humanized antibody may be made lessimmunogenic.

The term “rare”, as used herein, indicates an amino acid occurring atthat position in less than about 20% but usually less than about 10% ofsequences in a representative sample of sequences, and the term“common”, as used herein, indicates an amino acid occurring in more thanabout 25% but usually more than about 50% of sequences in arepresentative sample. For example, all human light and heavy chainvariable region sequences are respectively grouped into “subgroups” ofsequences that are especially homologous to each other and have the sameamino acids at certain critical positions (Kabat et al., supra). Whendeciding whether an amino acid in a human acceptor sequence is “rare” or“common” among human sequences, it will often be preferable to consideronly those human sequences in the same subgroup as the acceptorsequence.

Additional candidates for substitution are acceptor human frameworkamino acids that would be identified as part of a CDR region under thealternative definition proposed by Chothia et al., supra. Additionalcandidates for substitution are acceptor human framework amino acidsthat would be identified as part of a CDR region under the AbM and/orcontact definitions. Notably, CDR1 in the variable heavy chain isdefined as including residues 26-32.

Additional candidates for substitution are acceptor framework residuesthat correspond to a rare or unusual donor framework residue. Rare orunusual donor framework residues are those that are rare or unusual (asdefined herein) for murine antibodies at that position. For murineantibodies, the subgroup can be determined according to Kabat andresidue positions identified which differ from the consensus. Thesedonor specific differences may point to somatic mutations in the murinesequence which enhance activity. Unusual residues that are predicted toaffect binding are retained, whereas residues predicted to beunimportant for binding can be substituted.

Additional candidates for substitution are non-germline residuesoccurring in an acceptor framework region. For example, when an acceptorantibody chain (i.e., a human antibody chain sharing significantsequence identity with the donor antibody chain) is aligned to agermline antibody chain (likewise sharing significant sequence identitywith the donor chain), residues not matching between acceptor chainframework and the germline chain framework can be substituted withcorresponding residues from the germline sequence.

Other than the specific amino acid substitutions discussed above, theframework regions of humanized immunoglobulins are usually substantiallyidentical, and more usually, identical to the framework regions of thehuman antibodies from which they were derived. Of course, many of theamino acids in the framework region make little or no directcontribution to the specificity or affinity of an antibody. Thus, manyindividual conservative substitutions of framework residues can betolerated without appreciable change of the specificity or affinity ofthe resulting humanized immunoglobulin. Thus, in one embodiment thevariable framework region of the humanized immunoglobulin shares atleast 85% sequence identity to a human variable framework regionsequence or consensus of such sequences. In another embodiment, thevariable framework region of the humanized immunoglobulin shares atleast 90%, preferably 95%, more preferably 96%, 97%, 98% or 99% sequenceidentity to a human variable framework region sequence or consensus ofsuch sequences. In general, however, such substitutions are undesirable.

The humanized antibodies preferably exhibit a specific binding affinityfor antigen of at least 10⁷, 10⁸, 10⁹ or 10¹⁰, 10¹¹, 10¹², 10¹³ M⁻¹.Usually the upper limit of binding affinity of the humanized antibodiesfor antigen is within a factor of three, four or five of that of thedonor immunoglobulin. Often the lower limit of binding affinity is alsowithin a factor of three, four or five of that of donor immunoglobulin.Alternatively, the binding affinity can be compared to that of ahumanized antibody having no substitutions (e.g., an antibody havingdonor CDRs and acceptor FRs, but no FR substitutions). In suchinstances, the binding of the optimized antibody (with substitutions) ispreferably at least two- to three-fold greater, or three- to four-foldgreater, than that of the unsubstituted antibody. For makingcomparisons, activity of the various antibodies can be determined, forexample, by BIACORE (i.e., surface plasmon resonance using unlabelledreagents) or competitive binding assays.

c. Production of 11K2 Humanized Antibodies

A preferred embodiment of the present invention features a humanizedantibody to MCPs, in particular, for use in therapeutic and/ordiagnostic methodologies described herein. A particularly preferredstarting material for production of humanized antibodies is 11K2. 11K2is a pan-MCP antibody and is specific for MCP-1, MCP-2 and MCP-3. 11K2has been shown to inhibit MCP-induced chemotaxis (see Examples 3 and 5).The cloning and sequencing of cDNA encoding the 11K2 antibody heavy andlight chains is described in Example 10.

Suitable human acceptor antibody sequences are identified by computercomparisons of the amino acid sequences of the mouse variable regionswith the sequences of known human antibodies. The comparison isperformed separately for heavy and light chains but the principles aresimilar for each. In particular, variable domains from human antibodieswhose framework sequences exhibit a high degree of sequence identitywith the murine VL and VH framework regions were identified by query ofthe Kabat Database using NCBI BLAST (publicly accessible through theNational Institutes of Health NCBI internet server) with the respectivemurine framework sequences. In one embodiment, acceptor sequencessharing greater that 50% sequence identity with murine donor sequencesare selected. Preferably, acceptor antibody sequences sharing 60%, 70%,80%, 90% or more are selected.

A computer comparison of 11K2 revealed that the 11K2 light chain showsthe greatest sequence identity to human light chains of subtype kappa 1,and that the 11K2 heavy chain shows greatest sequence identity to humanheavy chains of subtype 1, as defined by Kabat et al., supra. Thus,light and heavy human framework regions are preferably derived fromhuman antibodies of these subtypes, or from consensus sequences of suchsubtypes. The preferred light chain human variable regions showinggreatest sequence identity to the corresponding region from 11K2 arefrom antibodies GI-486875 (Griffiths et al. (1993) EMBO J. 12(2),725-734). The preferred heavy chain human variable regions showinggreatest sequence identity to the corresponding region from 11K2 arefrom antibodies having Kabat ID Number 0000554 (Kipps and Duffy (1991),J. Clin. Invest. 87 (6): 2087-2096).

Residues are next selected for substitution, as follows. When an aminoacid differs between a 11K2 variable framework region and an equivalenthuman variable framework region, the human framework amino acid shouldusually be substituted by the equivalent mouse amino acid if it isreasonably expected that the amino acid:

noncovalently binds antigen directly,

is adjacent to a CDR region, is part of a CDR region under thealternative definition proposed by Chothia et al., supra, or otherwiseinteracts with a CDR region (e.g., is within about 3 of a CDR region)(e.g. amino acids at positions H29, H73, L49 of 11K2), or

participates in the VL-VH interface

Computer modeling of the 11K2 antibody heavy and light chain variableregions, and humanization of the 11K2 antibody is described in Example14. Briefly, a three-dimensional model was generated based on theclosest solved murine antibody structures for the heavy and lightchains. For this purpose, an antibody designated 184.1 (Protein DataBank (PDB) ID: 184.1) was chosen as a template for modeling the 11K2light chain, and an antibody designated E8 (PDB ID: 1OPG) was chosen asthe template to for modeling the heavy chain. The model was furtherrefined by a series of energy minimization steps to relieve unfavorableatomic contacts and optimize electrostatic and van der Wallsinteractions.

Three-dimensional structural information for the antibodies describedherein is publicly available, for example, from the ResearchCollaboratory for Structural Bioinformatics' Protein Data Bank (PDB).The PDB is freely accessible via the World Wide Web Internet and isdescribed by Berman et al. (2000) Nucleic Acids Research, 28:235.Computer modeling allows for the identification of CDR-interactingresidues. The computer model of the structure of 11K2 can in turn serveas a starting point for predicting the three-dimensional structure of anantibody containing the 11K2 complementarity determining regionssubstituted in human framework structures. Additional models can beconstructed representing the structure as further amino acidsubstitutions are introduced.

In general, substitution of one, most or all of the amino acidsfulfilling the above criteria is desirable. Accordingly, the humanizedantibodies of the present invention will usually contain a substitutionof a human light chain framework residue with a corresponding 11K2residue in at least 1, 2, and more usually 3, of the followingpositions: L49, L69 and L71. The humanized antibodies also usuallycontain a substitution of a human heavy chain framework residue with acorresponding 11K2 residue in at least 1, 2, 3, 4, 5, 6, and sometimes7, of the following positions: H27, H28, H29, H30, H48, H677, and H73.

In one embodiment, the humanized antibodies of the invention are basedon two versions of the humanised 11K2 variable heavy chain (H1 and H2)and two versions of the humanized 11K2 variable light chain (L1 and L2).The humanized antibody of the invention is based on any combination ofthese heavy and light chains (e.g. H1-L1, H1-L2, H2-L1, H2-L2). Version1 of the humanized heavy and light chains contains most backmutations(i.e. L49, L69, L71, H27, H28, H29, H30, H48, H67, and H73), whileversion 2 contains the fewest (i.e. L49, L71, H27, H29, and H73). Thesequence of humanized 11K2 variable heavy chain version 1 is set forthas SEQ ID NO: 47, and version 2 is set forth as SEQ ID NO: 48. Thesequence of humanized 11K2 variable light chain version 1 is set forthas SEQ ID NO: 49, and version 2 is set forth as SEQ ID NO: 50.

In one embodiment, the humanized antibody of the invention contains aheavy chain comprising SEQ ID NO: 47 and a light chain comprising SEQ IDNO: 49. In another embodiment, the humanized antibody of the inventioncontains a heavy chain comprising SEQ ID NO: 47 and a light chaincomprising SEQ ID NO: 50. In yet another embodiment, the humanizedantibody of the invention contains a heavy chain comprising SEQ ID NO:48 and a light chain comprising SEQ ID NO: 49. IN still anotherembodiment, the humanized antibody of the invention contains a heavychain comprising SEQ ID NO: 48 and a light chain comprising SEQ ID NO:50.

Occasionally, however, there is some ambiguity about whether aparticular amino acid meets the above criteria, and alternative variantimmunoglobulins are produced, one of which has that particularsubstitution, the other of which does not. In instances wheresubstitution with a murine residue would introduce a residue that israre in human immunoglobulins at a particular position, it may bedesirable to test the antibody for activity with or without theparticular substitution. If activity (e.g., binding affinity and/orbinding specificity) is about the same with or without the substitution,the antibody without substitution may be preferred, as it would beexpected to elicit less of a HAHA response, as described herein.

Other candidates for substitution are acceptor human framework aminoacids that are unusual for a human immunoglobulin at that position.These amino acids can be substituted with amino acids from theequivalent position of more typical human immunoglobulins.Alternatively, amino acids from equivalent positions in the mouse 11K2can be introduced into the human framework regions when such amino acidsare typical of human immunoglobulin at the equivalent positions.

In additional embodiments, when the human light chain framework acceptorimmunoglobulin is GI-486875, the light chain contains substitutions inat least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more usually 11, of thefollowing positions: L8, L11, L13, L15, L42, L45, L74, L76, L80, L83, orL104. In additional embodiments when the human heavy chain frameworkacceptor immunoglobulin is Kabat ID Number 000054, the heavy chaincontains substitutions in at least 1, 2, 3, 4, 5, 6, 7, 8; 9, 10, 11,12, 13, 14, 15, 16, 17, or more usually 18, of the following positions:H1, H5, H11, H12, H14, H16, 1120, H23, H38, H40, H42, H66, H75, H76,H80, H81, H82C, or H83. These positions are substituted with the aminoacid from the equivalent position of a human immunoglobulin having amore typical amino acid residue. Examples of appropriate amino acids tosubstitute are shown in FIG. 11. Table 1 summarizes the sequenceanalysis of the 11K2 VH and VL regions.

TABLE 1 Summary of 11K2 V-region sequence Chain Heavy Light Mousesubgroup 2C kappa 5 Human subgroup 1 kappa 1 Chothia canonical H1: 5residues, no class L1: 11 residues, class 2 CDR groupings H2: 17residues, class 2 L2: 7 residues, class 1 H3: 8 residues, no class L3: 9residues, class 1 Closest solved E8 184.1 mouse structure

Kabat ID sequences referenced herein are publicly available, forexample, from the Northwestern University Biomedical EngineeringDepartment's Kabat Database of Sequences of Proteins of ImmunologicalInterest. Three-dimensional structural information for antibodiesdescribed herein is publicly available, for example, from the ResearchCollaboratory for Structural Bioinformatics' Protein Data Bank (PDB).The PDB is freely accessible via the World Wide Web internet and isdescribed by Berman et al. (2000) Nucleic Acids Research, p 235-242.Germline gene sequences referenced herein are publicly available, forexample, from the National Center for Biotechnology Information (NCBI)database of sequences in collections of Igh, Ig kappa and Ig lambdagermline V genes (as a division of the National Library of Medicine(NLM) at the National Institutes of Health (NM)). Homology searching ofthe NCBI “Ig Germline Genes” database is provided by IgG BLAST™.

d. Production of 1A1 Humanized Antibodies

Another preferred embodiment of the present invention features ahumanized antibody to MCPs, in particular, for use in therapeutic and/ordiagnostic methodologies described herein, wherein the starting materialfor production of humanized antibodies is 1A1. 1A1 is a pan-MCPantibody, and is specific for MCP-1, MCP-2 and MCP-3. 1A1 has been shownto inhibit MCP-induced chemotaxis (see Examples 3 and 5). The cloningand sequencing of cDNA encoding the 1A1 antibody heavy and light chainsis described in Example 9.

Suitable human acceptor antibody sequences are identified by computercomparisons of the amino acid sequences of the mouse variable regionswith the sequences of known human antibodies. The comparison isperformed separately for heavy and light chains but the principles aresimilar for each. In particular, variable domains from human antibodieswhose framework sequences exhibit a high degree of sequence identitywith the murine VL and VH framework regions were identified by query ofthe Kabat Database using NCBI BLAST (publicly accessible through theNational Institutes of Health NCBI interne server) with the respectivemurine framework sequences. In one embodiment, acceptor sequencessharing greater that 50% sequence identity with murine donor sequencesare selected. Preferably, acceptor antibody sequences sharing 60%, 70%,80%, 90% or more are selected.

A computer comparison of 1A1 revealed that the 1A1 light chain is amember of mouse subgroup kappa 2 and shows the greatest sequenceidentity to human light chains of subtype kappa 2. The comparison alsorevealed that the 1A1 heavy chain is a member of mouse subgroup 2C andshows greatest sequence identity to human heavy chains of subgroup 1, asdefined by Kabat et al., supra. Thus, light and heavy human frameworkregions are preferably derived from human antibodies of these subtypes,or from consensus sequences of such subtypes. The preferred light chainhuman variable regions showing greatest sequence identity to thecorresponding region from 1A1 are from antibodies GI-284256 (Kennedy etal. (1991) J. Exp. Med. 173(4), 1033-1036). The preferred heavy chainhuman variable regions showing greatest sequence identity to thecorresponding region from 1A1 are from antibodies having Kabat ID Number037655 (Bejeck et al. (1995), Cancer Res. 55(11): 2346-2351).

Residues are next selected for substitution, as follows. When an aminoacid differs between a 1A1 variable framework region and an equivalenthuman variable framework region, the human framework amino acid shouldusually be substituted by the equivalent mouse amino acid if it isreasonably expected that the amino acid:

-   -   (1) noncovalently binds antigen directly,    -   (2) is adjacent to a CDR region, is part of a CDR region under        the alternative definition proposed by Chothia et al., supra, or        otherwise interacts with a CDR region (e.g., is within about 3        of a CDR region) (e.g. amino acids at positions H30, H73, or H93        of 1A1), or    -   (3) participates in the VL-VH interface (e.g. amino acids at        positions L36 and H91 of 1A1).

Computer modeling of the 1A1 antibody heavy and light chain variableregions, and humanization of the 1A1 antibody is described in theExamples. Briefly, a three-dimensional model was generated based on theclosest solved murine antibody structures for the heavy and lightchains. For this purpose, an antibody designated Fab1583 (1NLD; 2.9 Å)and D2.5 (1YEE; 2.2 Å) were chosen as templates for modeling the 1A1light chain, and antibodies designated 2E8 (12E8; 1.9 Å) and F9.13.7(1FB1; 3.0 Å) were chosen as templates for modeling the heavy chain. Themodel was further refined by a series of energy minimization steps torelieve unfavorable atomic contacts and optimize electrostatic and vander Walls interactions.

Three-dimensional structural information for the antibodies describedherein is publicly available, for example, from the ResearchCollaboratory for Structural Bioinformatics' Protein Data Bank (PDB).The PDB is freely accessible via the World Wide Web internet and isdescribed by Berman et al. (2000) Nucleic Acids Research, 28:235.Computer modeling allows for the identification of CDR-interactingresidues. The computer model of the structure of 1A1 can in turn serveas a starting point for predicting the three-dimensional structure of anantibody containing the 1A1 complementarity determining regionssubstituted in human framework structures. Additional models can beconstructed representing the structure as further amino acidsubstitutions are introduced.

In general, substitution of one, most or all of the amino acidsfulfilling the above criteria is desirable. Accordingly, the humanizedantibodies of the present invention will usually contain a substitutionof a human light chain framework residue with a corresponding 1A1residue in at least 1, 2 and sometimes 3, of the following positions:L2, L36, and L45. The humanized antibodies also usually contain asubstitution of a human heavy chain framework residue with acorresponding 1A1 residue in at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, andsometimes 11, of the following positions: H27, H28, H29, H30, H66, H69,H73, H76, H91, H93, and H94.

In one embodiment, the humanized antibodies of the invention are basedon two versions of the humanized 1A1 variable heavy chain (H1 and H2)and two versions of the humanized 1A1 variable light chain (L1 and L2).The humanized antibody of invention is based on any combination of the1A1 humanized heavy and light chains (e.g. H1-L1, H1-L2, H2-L1, H2-L2).Version 1 of the humanized heavy and light chains contains mostbackmutations (i.e. L2, L36, L45, H27, H28, H29, H30, H66, H69, H73,H76, H91, H93, and H94), while version 2 contains the fewest (i.e. L2,L36, H29, H30, H73, H91, H93, and H94). The sequence of humanized 1A1variable heavy chain version 1 is set forth as SEQ ID NO: 53, andversion 2 is set forth as SEQ ID NO: 54. The sequence of humanized 1A1variable light chain version 1 is set forth as SEQ ID NO: 55, andversion 2 is set forth as SEQ ID NO: 56.

In one embodiment, the humanized antibody of the invention contains aheavy chain comprising SEQ ID NO: 53 and a light chain comprising SEQ IDNO: 55. In another embodiment, the humanized antibody of the inventioncontains a heavy chain comprising SEQ ID NO: 53 and a light chaincomprising SEQ ID NO: 56. In yet another embodiment, the humanizedantibody of the invention contains a heavy chain comprising SEQ ID NO:54 and a light chain comprising SEQ ID NO: 55. In still anotherembodiment, the humanized antibody of the invention contains a heavychain comprising SEQ ID NO: 554 and a light chain comprising SEQ ID NO:56.

Occasionally; however, there is some ambiguity about whether aparticular amino acid meets the above criteria, and alternative variantimmunoglobulins are produced, one of which has that particularsubstitution, the other of which does not. In instances wheresubstitution with a murine residue would introduce a residue that israre in human immunoglobulins at a particular position, it may bedesirable to test the antibody for activity with or without theparticular substitution. If activity (e.g., binding affinity and/orbinding specificity) is about the same with or without the substitution,the antibody without substitution may be preferred, as it would beexpected to elicit less of a HAHA response, as described herein.

Other candidates for substitution are acceptor human framework aminoacids that are unusual for a human immunoglobulin at that position.These amino acids can be substituted with amino acids from theequivalent position of more typical human immunoglobulins.Alternatively, amino acids from equivalent positions in the mouse 11K2can be introduced into the human framework regions when such amino acidsare typical of human immunoglobulin at the equivalent positions.

In additional embodiments, when the human light chain framework acceptorimmunoglobulin is GI-284256, the light chain contains substitutions inat least 1, 2, 3, 4, 5, 6, 7, 8, or more usually 9, of the followingpositions: L3, L8, L9, L11, L12, L14, L37, L63, or L99. In additionalembodiments when the human heavy chain framework acceptor immunoglobulinis Kabat ID Number 037655, the heavy chain contains substitutions in atleast 1, 2, 3, 4, 5, 6, 7, 8, or more usually 9, of the followingpositions: H1, H6, H14, H16, H23, H42, H80, H82B, or H87. Thesepositions are substituted with the amino acid from the equivalentposition of a human immunoglobulin having a more typical amino acidresidue. Examples of appropriate amino acids to substitute are shown inFIG. 11. Table 2 summarizes the sequence analysis of the 1A1 VH and VLregions.

TABLE 2 Summary of 1A1 V-region sequence Chain Heavy Light Mousesubgroup 2C kappa 2 Human subgroup 1 kappa 2 Chothia canonical H1: 5residues, class 1 L1: 16 residues, class 4 CDR groupings H2: 17residues, class 2 L2: 7 residues, class 1 H3: 13 residues, no class L3:9 residues, class 1 Closest solved 2E8 (12E8; 1.9 Å) Fab1583 (1NLD; 2.9Å) mouse structure F9.13.7 (1FB1; 3.0 Å D2.5 (1YEE; 2.2 Å)

Kabat ID sequences referenced herein are publicly available, forexample, from the Northwestern University Biomedical EngineeringDepartment's Kabat Database of Sequences of Proteins of ImmunologicalInterest. Three-dimensional structural information for antibodiesdescribed herein is publicly available, for example, from the ResearchCollaboratory for Structural Bioinformatics' Protein Data Bank (PDB).The PDB is freely accessible via the World Wide Web internet and isdescribed by Berman et al. (2000) Nucleic Acids Research, p 235-242.Germline gene sequences referenced herein are publicly available, forexample, from the National Center for Biotechnology Information (NCBI)database of sequences in collections of Igh, Ig kappa and Ig lambdagermline V genes (as a division of the National Library of Medicine(NLM) at the National Institutes of Health (NIH)). Homology searching ofthe NCBI “Ig Germline Genes” database is provided by IgG BLAST™.

In another embodiment, a humanized antibody of the present invention hasstructural features, as described herein, and specifically binds to anepitope comprising MCP-1 and MCP-2.

3. Human Antibodies

Human antibodies against MCPs are provided by a variety of techniquesdescribed below. Some human antibodies are selected by competitivebinding experiments, or otherwise, to have the same epitope specificityas a particular mouse antibody, such as one of the mouse monoclonalsdescribed herein. Human antibodies can also be screened for a particularepitope specificity by using only a fragment of an MCP molecule as theimmunogen, and/or by screening antibodies against a collection ofdeletion mutants of MCP. Human antibodies preferably have human IgG1isotype specificity.

a. Trioma Methodology

The basic approach and an exemplary cell fusion partner, SPAZ-4, for usein this approach have been described by Oestberg et al., Hybridoma 2:361(1983); Oestberg, U.S. Pat. No. 4,634,664; and Engleman et al., U.S.Pat. No. 4,634,666 (each of which is incorporated by reference in itsentirety for all purposes). The antibody-producing cell lines obtainedby this method are called triomas, because they are descended from threecells; two human and one mouse. Initially, a mouse myeloma line is fusedwith a human B-lymphocyte to obtain a non-antibody-producing xenogeneichybrid cell, such as the SPAZ-4 cell line described by Oestberg, supra.The xenogeneic cell is then fused with an immunized human B-lymphocyteto obtain an antibody-producing trioma cell line. Triomas have beenfound to produce antibody more stably than ordinary hybridomas made fromhuman cells.

The immunized B-lymphocytes are obtained from the blood, spleen, lymphnodes or bone marrow of a human donor. If antibodies against a specificantigen or epitope are desired, it is preferable to use that antigen orepitope thereof for immunization. Immunization can be either in vivo orin vitro. For example, for in vivo B cells are typically isolated from ahuman immunized with MCP-1, a fragment thereof, larger polypeptidecontaining MCP-1 or fragment, or an anti-idiotypic antibody to anantibody to MCP-1. In some methods, B cells are isolated from the samesubject who is ultimately to be administered antibody therapy. For invitro immunization, B-lymphocytes are typically exposed to antigen for aperiod of 7-14 days in a media such as RPMI-1640 (see Engleman, supra)supplemented with 10% human plasma.

The immunized B-lymphocytes are fused to a xenogeneic hybrid cell suchas SPAZ-4 by well-known methods. For example, the cells are treated with40-50% polyethylene glycol of MW 1000-4000, at about 37 degrees C., forabout 5-10 min. Cells are separated from the fusion mixture andpropagated in media selective for the desired hybrids (e.g., HAT or AH).Clones secreting antibodies having the required binding specificity areidentified by assaying the trioma culture medium for the ability to bindto Aβ or a fragment thereof. Triomas producing human antibodies havingthe desired specificity are subcloned by the limiting dilution techniqueand grown in vitro in culture medium. The trioma cell lines obtained arethen tested for the ability to bind Aβ or a fragment thereof.

Although triomas are genetically stable they do not produce antibodiesat very high levels. Expression levels can be increased by cloningantibody genes from the trioma into one or more expression vectors, andtransforming the vector into standard mammalian, bacterial or yeast celllines.

b. Transgenic Non-Human Mammals

Human antibodies against MCPs can also be produced from non-humantransgenic mammals having transgenes encoding at least a segment of thehuman immunoglobulin locus. Usually, the endogenous immunoglobulin locusof such transgenic mammals is functionally inactivated. Preferably, thesegment of the human immunoglobulin locus includes unrearrangedsequences of heavy and light chain components. Both inactivation ofendogenous immunoglobulin genes and introduction of exogenousimmunoglobulin genes can be achieved by targeted homologousrecombination, or by introduction of YAC chromosomes. The transgenicmammals resulting from this process are capable of functionallyrearranging the immunoglobulin component sequences, and expressing arepertoire of antibodies of various isotypes encoded by humanimmunoglobulin genes, without expressing endogenous immunoglobulingenes. The production and properties of mammals having these propertiesare described in detail by, e.g., Lonberg et al., WO93/12227 (1993);U.S. Pat. No. 5,877,397, U.S. Pat. No. 5,874,299, U.S. Pat. No.5,814,318, U.S. Pat. No. 5,789,650, U.S. Pat. No. 5,770,429, U.S. Pat.No. 5,661,016, U.S. Pat. No. 5,633,425, U.S. Pat. No. 5,625,126, U.S.Pat. No. 5,569,825, U.S. Pat. No. 5,545,806, Nature 148:1547 (1994),Nature Biotechnology 14:826 (1996), Kucherlapati, WO 91/10741 (1991)(each of which is incorporated by reference in its entirety for allpurposes). Transgenic mice are particularly suitable. For example,anti-MCP-1 antibodies are obtained by immunizing a transgenic nonhumanmammal, such as described by Lonberg or Kucherlapati, supra, with MCP-1or a fragment thereof. Monoclonal antibodies are prepared by, e.g.,fusing B-cells from such mammals to suitable myeloma cell lines usingconventional Kohler-Milstein technology. Human polyclonal antibodies canalso be provided in the form of serum from humans immunized with animmunogenic agent. Optionally, such polyclonal antibodies can beconcentrated by affinity purification using MCP-1 or other MCP peptideas an affinity reagent

c. Phage Display Methods

A further approach for obtaining human anti-MCP antibodies is to screena DNA library from human B cells according to the general protocoloutlined by Huse et al., Science 246:1275-1281 (1989). As described fortrioma methodology, such B cells can be obtained from a human immunizedwith MCP-1, fragments, longer polypeptides containing MCP-1 or fragmentsor anti-idiotypic antibodies. Optionally, such B cells are obtained froma subject who is ultimately to receive antibody treatment. Antibodiesbinding to MCP-1 or a fragment thereof are selected. Sequences encodingsuch antibodies (or a binding fragments) are then cloned and amplified.The protocol described by Huse is rendered more efficient in combinationwith phage-display technology. See, e.g., Dower et al., WO 91/17271,McCafferty et al., WO 92/01047, Herzig et al., U.S. Pat. No. 5,877,218,Winter et al., U.S. Pat. No. 5,871,907, Winter et al., U.S. Pat. No.5,858,657, Holliger et al., U.S. Pat. No. 5,837,242, Johnson et al.,U.S. Pat. No. 5,733,743 and Hoogenboom et al., U.S. Pat. No. 5,565,332(each of which is incorporated by reference in its entirety for allpurposes). In these methods, libraries of phage are produced in whichmembers display different antibodies on their outer surfaces. Antibodiesare usually displayed as Fv or Fab fragments. Phage displayingantibodies with a desired specificity are selected by affinityenrichment to au MCP-1 peptide or fragment thereof.

In a variation of the phage-display method, human antibodies having thebinding specificity of a selected murine antibody can be produced. SeeWinter, WO 92/20791. In this method, either the heavy or light chainvariable region of the selected murine antibody is used as a startingmaterial. It for example, a light chain variable region is selected asthe starting material, a phage library is constructed in which membersdisplay the same light chain variable region (i.e., the murine startingmaterial) and a different heavy chain variable region. The heavy chainvariable regions are obtained from a library of rearranged human heavychain variable regions. A phage showing strong specific binding forMCP-1, MCP-2, MCP-3, or a combination thereof (e.g., at least 10⁸ andpreferably at least 10⁹ M⁻¹) is selected. The human heavy chain variableregion from this phage then serves as a starting material forconstructing a further phage library. In this library, each phagedisplays the same heavy chain variable region (i.e., the regionidentified from the first display library) and a different light chainvariable region. The light chain variable regions are obtained from alibrary of rearranged human variable light chain regions. Again, phageshowing strong specific binding for MCP-1, MCP-2, MCP-3, or acombination thereof, are selected. These phage display the variableregions of completely human anti-MCP antibodies. These antibodiesusually have the same or similar epitope specificity as the murinestarting material.

4. Production of Variable Regions

Having conceptually selected the CDR and framework components ofhumanized immunoglobulins, a variety of methods are available forproducing such immunoglobulins. Because of the degeneracy of the code, avariety of nucleic acid sequences will encode each immunoglobulin aminoacid sequence. The desired nucleic acid sequences can be produced by denovo solid-phase DNA synthesis or by PCR mutagenesis of an earlierprepared variant of the desired polynucleotide. Oligonucleotide-mediatedmutagenesis is a preferred method for preparing substitution, deletionand insertion variants of target polypeptide DNA. See Adelman et al.,DNA 2:183 (1983). Briefly, the target polypeptide DNA is altered byhybridizing an oligonucleotide encoding the desired mutation to asingle-stranded DNA template. After hybridization, a DNA polymerase isused to synthesize an entire second complementary strand of the templatethat incorporates the oligonucleotide primer, and encodes the selectedalteration in the target polypeptide DNA.

5. Selection of Constant Regions

The variable segments of antibodies produced as described supra (e.g.,the heavy and light chain variable regions of chimeric, humanized, orhuman antibodies) are typically linked to at least a portion of animmunoglobulin constant region (Fe), typically that of a humanimmunoglobulin. Human constant region DNA sequences can be isolated inaccordance with well known procedures from a variety of human cells, butpreferably immortalized B cells (see Kabat et al., supra, and Liu etal., WO87/02671) (each of which is incorporated by reference in itsentirety for all purposes). Ordinarily, the antibody will contain bothlight chain and heavy chain constant regions. The heavy chain constantregion usually includes CHI, hinge, CH2, CH3, and CH4 regions. Theantibodies described herein include antibodies having all types ofconstant regions, including IgM, IgG, IgD, IgA and IgE, and any isotype,including IgG1, IgG2, IgG3 and IgG4. The choice of constant regiondepends, in part, whether antibody-dependent complement and/or cellularmediated toxicity is desired. For example, isotopes IgG1 and IgG3 havecomplement activity and isotypes IgG2 and IgG4 do not. When it isdesired that the antibody (e.g., humanized antibody) exhibit cytotoxicactivity, the constant domain is usually a complement fixing constantdomain and the class is typically IgG1. When such cytotoxic activity isnot desirable, the constant domain may be of the IgG2 class. Choice ofisotype can also affect passage of antibody into the brain. Humanisotype IgG1 is preferred. Light chain constant regions can be lambda orkappa. The humanized antibody may comprise sequences from more than oneclass or isotype. Antibodies can be expressed as tetramers containingtwo light and two heavy chains, as separate heavy chains, light chains,as Fab, Fab′ F(ab)₂, and Fv, or as single chain antibodies in whichheavy and light chain variable domains are linked through a spacer.

6. Chemical Modifications

In some embodiments, the antibodies and antibody fragments of theinvention may be chemically modified to provide a desired effect. Forexample, pegylation of antibodies and antibody fragments of theinvention may be carried out by any of the pegylation reactions known inthe art, as described, for example, in the following references: Focuson Growth Factors 3:4-10 (1992); EP 0 154 316; and EP 0 401 384 (each ofwhich is incorporated by reference herein in its entirety). Preferably,the pegylation is carried out via an acylation reaction or an alkylationreaction with a reactive polyethylene glycol molecule (or an analogousreactive water-soluble polymer). A preferred water-soluble polymer forpegylation of the antibodies and antibody fragments of the invention ispolyethylene glycol (PEG). As used herein, “polyethylene glycol” ismeant to encompass any of the forms of PEG that have been used toderivatize other proteins, such as mono (Cl—ClO) alkoxy- oraryloxy-polyethylene glycol.

Methods for preparing pegylated antibodies and antibody fragments of theinvention will generally comprise the steps of (a) reacting the antibodyor antibody fragment with polyethylene glycol, such as a reactive esteror aldehyde derivative of PEG, under conditions whereby the antibody orantibody fragment becomes attached to one or more PEG groups, and (b)obtaining the reaction products. It will be apparent to one of ordinaryskill in the art to select the optimal reaction conditions or theacylation reactions based on known parameters and the desired result.

Pegylated antibodies and antibody fragments may generally be used totreat conditions that may be alleviated or modulated by administrationof the antibodies and antibody fragments described herein. Generally thepegylated antibodies and antibody fragments have increased half-life, ascompared to the nonpegylated antibodies and antibody fragments. Thepegylated antibodies and antibody fragments may be employed alone,together, or in combination with other pharmaceutical compositions. Inone embodiment, the invention describes pegylated Fab antibodies,including pegylated humanized Fab-11K2 and pegylated murine Fab-11K2.

In other embodiments of the invention the antibodies or antigen-bindingfragments thereof are conjugated to albumen using art recognizedtechniques.

In another embodiment of the invention, antibodies, or fragmentsthereof, are modified to reduce or eliminate potential glycosylationsites. Such modified antibodies are often referred to as “aglycosylated”antibodies. In order to improve the binding affinity of an antibody orantigen-binding fragment thereof, glycosylation sites of the antibodycan be altered, for example, by mutagenesis (e.g., site-directedmutagenesis). “Glycosylation sites” refer to amino acid residues whichare recognized by a eukaryotic cell as locations for the attachment ofsugar residues. The amino acids where carbohydrate, such asoligosaccharide, is attached are typically asparagine (N-linkage),serine (O-linkage), and threonine (O-linkage) residues. In order toidentify potential glycosylation sites within an antibody orantigen-binding fragment, the sequence of the antibody is examined, forexample, by using publicly available databases such as the websiteprovided by the Center for Biological Sequence Analysis (seehttp://www.cbs.dtu.dk/services/NetNGlyc/ for predicting N-linkedglycoslyation sites) and http://www.cbs.dtu.dk/services/NetOGlyc/ forpredicting O-linked glycoslyation sites). Additional methods foraltering glycosylation sites of antibodies are described in U.S. Pat.Nos. 6,350,861 and 5,714,350.

In yet another embodiment of the invention, antibodies or fragmentsthereof can be altered wherein the constant region of the antibody ismodified to reduce at least one constant region-mediated biologicaleffector function relative to an unmodified antibody. To modify anantibody of the invention such that it exhibits reduced binding to theFc receptor (FcR), the immunoglobulin constant region segment of theantibody can be mutated at particular regions necessary for FcRinteractions (see e.g., Canfield, S. M. and S. L. Morrison (1991) J.Exp. Med. 173:1483-1491; and Lund, J. et al. (1991) J. of Immunol.147:2657-2662). Reduction in FcR binding ability of the antibody mayalso reduce other effector functions which rely on FcR interactions,such as opsonization and phagocytosis and antigen-dependent cellularcytotoxicity.

In a particular embodiment the invention further features antibodieshaving altered effector function, such as the ability to bind effectormolecules, for example, complement or a receptor on an effector cell. Inparticular, the humanized antibodies of the invention have an alteredconstant region, e.g., Fc region, wherein at least one amino acidresidue in the Fc region has been replaced with a different residue orside chain thereby reducing the ability of the antibody to bind the FcR.Reduction in FcR binding ability of the antibody may also reduce othereffector functions which rely on FcR interactions, such as opsonizationand phagocytosis and antigen-dependent cellular cytotoxicity. In oneembodiment, the modified humanized antibody is of the IgG class,comprises at least one amino acid residue replacement in the Fc regionsuch that the humanized antibody has an altered effector function, e.g.,as compared with an unmodified humanized antibody. In particularembodiments, the humanized antibody of the invention has an alteredeffector function such that it is less immunogenic (e.g., does notprovoke undesired effector cell activity, lysis, or complement binding),and/or has a more desirable half-life while retaining specificity forMCP1, MCP-2, and/or MCP-3.

Alternatively, the invention features humanized antibodies havingaltered constant regions to enhance FcR binding, e.g., FcγR3 binding.Such antibodies are useful for modulating effector cell function, e.g.,for increasing ADCC activity, e.g., particularly for use in oncologyapplications.

As used herein, “Antibody-dependent cell-mediated cytotoxicity” and“ADCC” refer to a cell-mediated reaction in which nonspecific cytotoxiccells that express FcRs (e.g. Natural Killer (NK) cells, neutrophils,and macrophages) recognize bound antibody on a target cell andsubsequently cause lysis of the target cell. The primary for mediatingADCC; NK cells, express FcγRIII only, whereas monocytes express FcγRI,FcγRII and FcγRIII. of the antibody, e.g., a conjugate of the antibodyand another agent or antibody.

7. Expression of Recombinant Antibodies

Chimeric, humanized and human antibodies are typically produced byrecombinant expression. Nucleic acids encoding humanized light and heavychain variable regions, optionally linked to constant regions, areinserted into expression vectors. The light and heavy chains can becloned in the same or different expression vectors. The DNA segmentsencoding immunoglobulin chains are operably linked to control sequencesin the expression vector(s) that ensure the expression of immunoglobulinpolypeptides. Expression control sequences include, but are not limitedto, promoters (e.g., naturally-associated or heterologous promoters),signal sequences, enhancer elements, and transcription terminationsequences. Preferably, the expression control sequences are eukaryoticpromoter systems in vectors capable of transforming or transfectingeukaryotic host cells. Once the vector has been incorporated into theappropriate host, the host is maintained under conditions suitable forhigh level expression of the nucleotide sequences, and the collectionand purification of the crossreacting antibodies.

These expression vectors are typically replicable in the host organismseither as episomes or as an integral part of the host chromosomal DNA.Commonly, expression vectors contain selection markers (e.g.,ampicillin-resistance, hygromycin-resistance, tetracycline resistance orneomycin resistance) to permit detection of those cells transformed withthe desired DNA sequences (see, e.g., Itakura et al., U.S. Pat. No.4,704,362).

E. coli is one prokaryotic host particularly useful for cloning thepolynucleotides (e.g., DNA sequences) of the present invention. Othermicrobial hosts suitable for use include bacilli, such as Bacillussubtilus, and other enterobacteriaceae, such as Salmonella, Serratia,and various Pseudomonas species. In these prokaryotic hosts, one canalso make expression vectors, which will typically contain expressioncontrol sequences compatible with the host cell (e.g., an origin ofreplication). In addition, any number of a variety of well-knownpromoters will be present, such as the lactose promoter system, atryptophan (trp) promoter system, a beta-lactamase promoter system, or apromoter system from phage lambda. The promoters will typically controlexpression, optionally with an operator sequence, and have ribosomebinding site sequences and the like, for initiating and completingtranscription and translation.

Other microbes, such as yeast, are also useful for expression.Saccharomyces is a preferred yeast host, with suitable vectors havingexpression control sequences (e.g., promoters), an origin ofreplication, termination sequences and the like as desired. Typicalpromoters include 3-phosphoglycerate kinase and other glycolyticenzymes. Inducible yeast promoters include, among others, promoters fromalcohol dehydrogenase, isocytochrome C, and enzymes responsible formaltose and galactose utilization.

In addition to microorganisms, mammalian tissue cell culture may also beused to express and produce the polypeptides of the present invention(e.g., polynucleotides encoding immunoglobulins or fragments thereof).See Winnacker, From Genes to Clones, VCH Publishers, N.Y., N.Y. (1987).Eukaryotic cells are actually preferred, because a number of suitablehost cell lines capable of secreting heterologous proteins (e.g., intactimmunoglobulins) have been developed in the art, and include CHO celllines, various Cos cell lines, HeLa cells, preferably, myeloma celllines, or transformed B-cells or hybridomas. Preferably, the cells arenonhuman. Expression vectors for these cells can include expressioncontrol sequences, such as an origin of replication, a promoter, and anenhancer (Queen et al., Immunol. Rev. 89:49 (1986)), and necessaryprocessing information sites, such as ribosome binding sites, RNA splicesites, polyadenylation sites, and transcriptional terminator sequences.Preferred expression control sequences are promoters derived fromimmunoglobulin genes, SV40, adenovirus, bovine papilloma virus,cytomegalovirus and the like. See Co et al., J. Immunol. 148:1149(1992).

Alternatively, antibody-coding sequences can be incorporated intransgenes for introduction into the genome of a transgenic animal andsubsequent expression in the milk of the transgenic animal (see, e.g.,Deboer et al., U.S. Pat. No. 5,741,957, Rosen, U.S. Pat. No. 5,304,489,and Meade et al., U.S. Pat. No. 5,849,992). Suitable transgenes includecoding sequences for light and/or heavy chains in operable linkage witha promoter and enhancer from a mammary gland specific gene, such ascasein or beta lactoglobulin.

The vectors containing the polynucleotide sequences of interest (e.g.,the heavy and light chain encoding sequences and expression controlsequences) can be transferred into the host cell by well-known methods,which vary depending on the type of cellular host. For example, calciumchloride transfection is commonly utilized for prokaryotic cells,whereas calcium phosphate treatment, electroporation, lipofection,biolistics or viral-based transfection may be used for other cellularhosts. (See generally Sambrook et al., Molecular Cloning: A LaboratoryManual (Cold Spring Harbor Press, 2nd ed., 1989) (incorporated byreference in its entirety for all purposes). Other methods used totransform mammalian cells include the use of polybrene, protoplastfusion, liposomes, electroporation, and microinjection (see generally,Sambrook et al., supra). For production of transgenic animals,transgenes can be microinjected into fertilized oocytes, or can beincorporated into the genome of embryonic stem cells, and the nuclei ofsuch cells transferred into enucleated oocytes.

When heavy and light chains are cloned on separate expression vectors,the vectors are co-transfected to obtain expression and assembly ofintact immunoglobulins. Once expressed, the whole antibodies, theirdimers, individual light and heavy chains, or other immunoglobulin formsof the present invention can be purified according to standardprocedures of the art, including ammonium sulfate precipitation,affinity columns, column chromatography, HPLC purification, gelelectrophoresis and the like (see generally Scopes, Protein Purification(Springer-Verlag, N.Y., (1982)). Substantially pure immunoglobulins ofat least about 90 to 95% homogeneity are preferred, and 98 to 99% ormore homogeneity most preferred, for pharmaceutical uses.

The invention also includes aglycosylated antibodies, which may bedesirable for therapeutic treatment of human disease. Humanizedantibodies with altered glycosylation are produced by expression of anucleic acid encoding a human antibody in a cell line that has alteredability to post-translationally modify polypeptides, e.g., glycosylatepolypeptides. For example, e.g, EP 1,176,195 describes afucosyltransferase mutant, WO 99/54342 describes a CHO cell lineengineered with regulatable GntIII expression resulting in increasedbisecting GlcNAc structures having enhanced effector function, Shieldset al. ((2002) J. Biol. Chem. 277 26733) describes a hypofucosylatedanti-HER2 hu4D5 mAb made in mutant lec13 cells has improved ADCC, andUmana et al. ((1999) Nat. Biotechnol. 17: 176) describes a mAb withaltered bisecting glycoforms made in cells overexpressing rat GnTII thatexhibits improved ADCC.

8. Antibody Fragments

Also contemplated within the scope of the instant invention are antibodyfragments. In one embodiment, fragments of non-human, chimeric and/orhuman antibodies are provided. In another embodiment, fragments ofhumanized antibodies are provided. Typically, these fragments exhibitspecific binding to antigen with an affinity of at least 10⁷, and moretypically 10⁸ or 10⁹M⁻¹. Humanized antibody fragments include separateheavy chains, light chains Fab, Fab′ F(ab′)2, Fabc, and Fv. Preferredfragments of the invention include humanized 11K2 Fab and humanized 1A1Fab antibodies. In yet another embodiment, the invention includes murine11K2 Fab fragments and 1A1 Fab fragments. Fragments are produced byrecombinant DNA techniques, or by enzymatic or chemical separation ofintact immunoglobulins.

B. Nucleic Acid Encoding Immunologic and Therapeutic Agents

Immune responses against MCPs can also be induced by administration ofnucleic acids encoding antibodies and their component chains used forpassive immunization. Such nucleic acids can be DNA or RNA. A nucleicacid segment encoding an immunogen is typically linked to regulatoryelements, such as a promoter and enhancer, that allow expression of theDNA segment in the intended target cells of a subject. For expression inblood cells, as is desirable for induction of an immune response,promoter and enhancer elements from light or heavy chain immunoglobulingenes or the CMV major intermediate early promoter and enhancer aresuitable to direct expression. The linked regulatory elements and codingsequences are often cloned into a vector. For administration ofdouble-chain antibodies, the two chains can be cloned in the same orseparate vectors.

A number of viral vector systems are available including retroviralsystems (see, e.g., Lawrie and Tumin, Cur. Opin. Genet. Develop.3:102-109 (1993)); adenoviral vectors (see, e.g., Bett et al., J. Virol.67:5911 (1993)); adeno-associated virus vectors (see, e.g., Zhou et al.,J. Exp. Med. 179:1867 (1994)), viral vectors from the pox familyincluding vaccinia virus and the avian pox viruses, viral vectors fromthe alpha virus genus such as those derived from Sindbis and SemlikiForest Viruses (see, e.g., Dubensky et al., J. Virol. 70:508 (1996)),Venezuelan equine encephalitis virus (see Johnston et al., U.S. Pat. No.5,643,576) and rhabdoviruses, such as vesicular stomatitis virus (seeRose, WO 96/34625) and papillomaviruses (Ohe et al., Human Gene Therapy6:325 (1995); Woo et al., WO 94/12629 and Xiao & Brandsma, NucleicAcids. Res. 24, 2630-2622 (1996)).

DNA encoding an immunogen, or a vector containing the same, can bepackaged into liposomes. Suitable lipids and related analogs aredescribed by Eppstein et al., U.S. Pat. No. 5,208,036, Feigner et al.,U.S. Pat. No. 5,264,618, Rose, U.S. Pat. No. 5,279,833, and Epand etal., U.S. Pat. No. 5,283,185. Vectors and DNA encoding an immunogen canalso be adsorbed to or associated with particulate carriers, examples ofwhich include polymethyl methacrylate polymers and polylactides and poly(lactide-co-glycolides), see, e.g., McGee et al., J. Micro Encap.(1996).

Gene therapy vectors or naked polypeptides (e.g., DNA) can be deliveredin vivo by administration to an individual subject, typically bysystemic administration (e.g., intravenous, intraperitoneal, nasal,gastric, intradermal, intramuscular, subdermal, or intracranialinfusion) or topical application (see e.g., Anderson et al., U.S. Pat.No. 5,399,346). The term “naked polynucleotide” refers to apolynucleotide not complexed with colloidal materials. Nakedpolynucleotides are sometimes cloned in a plasmid vector. Such vectorscan further include facilitating agents such as bupivacine (Attardo etal., U.S. Pat. No. 5,593,970). DNA can also be administered using a genegun. See Xiao & Brandsma, supra. The DNA encoding an immunogen isprecipitated onto the surface of microscopic metal beads. Themicroprojectiles are accelerated with a shock wave or expanding heliumgas, and penetrate tissues to a depth of several cell layers. Forexample, The Accel™ Gene Delivery Device manufactured by Agacetus, Inc.Middleton Wis. is suitable. Alternatively, naked DNA can pass throughskin into the blood stream simply by spotting the DNA onto skin withchemical or mechanical irritation (see Howell et al., WO 95/05853).

In a further variation, vectors encoding immunogens can be delivered tocells ex vivo, such as cells explanted from an individual subject (e.g.,lymphocytes, bone marrow aspirates, tissue biopsy) or universal donorhematopoietic stem cells, followed by reimplantation of the cells into asubject, usually after selection for cells which have incorporated thevector.

II. Prophylactic and Therapeutic Methods

The present invention is directed inter alia to treatment of diseasesassociated with MCP-associated inflammation, by administration oftherapeutic immunological reagents (e.g., humanized immunoglobulins) tospecific epitopes within an MCP protein to a subject under conditionsthat generate a beneficial therapeutic response in a subject, forexample, for the prevention or treatment of a disorder associated withdetrimental MCP activity. The invention is also directed to use of thedisclosed immunological reagents (e.g., humanized immunoglobulins) inthe manufacture of a medicament for the treatment or prevention of anMCP-associated disease.

The term “treatment” as used herein, is defined as the application oradministration of a therapeutic agent to a subject, or application oradministration of a therapeutic agent to an isolated tissue or cell linefrom a subject, who has a disease, a symptom of disease or apredisposition toward a disease, with the purpose to cure, heal,alleviate, relieve, alter, remedy, ameliorate, improve or affect thedisease, the symptoms of disease or the predisposition toward disease.

Therapeutic agents of the invention are typically substantially purefrom undesired contaminant. This means that an agent is typically atleast about 50% w/w (weight/weight) purity, as well as beingsubstantially free from interfering proteins and contaminants. Sometimesthe agents are at least about 80% w/w and, more preferably at least 90or about 95% w/w purity. However, using conventional proteinpurification techniques, homogeneous peptides of at least 99% w/w can beobtained.

The methods can be used on both asymptomatic subjects and thosecurrently showing symptoms of disease. The antibodies used in suchmethods can be human, humanized, chimeric or nonhuman antibodies, orfragments thereof (e.g., antigen binding fragments) and can bemonoclonal or polyclonal, as described herein. In yet another aspect,the invention features administering antibodies prepared from a humanimmunized with an MCP peptide, which human can be the subject to betreated with antibody.

In another aspect, the invention features administering an antibody witha pharmaceutical carrier as a pharmaceutical composition. Alternatively,the antibody can be administered to a subject by administering apolynucleotide encoding at least one antibody chain. The polynucleotideis expressed to produce the antibody chain in the subject. Optionally,the polynucleotide encodes heavy and light chains of the antibody. Thepolynucleotide is expressed to produce the heavy and light chains in thesubject. In exemplary embodiments, the subject is monitored for level ofadministered antibody in the blood of the subject.

The invention thus fulfills a longstanding need for therapeutic regimesfor preventing or ameliorating inflammation associated with MCPs.

A. Disorders Amenable to Treatment

As used herein, the terms “a disorder in which MCP activity isdetrimental” and “an MCP-associated disorder” are intended to includediseases and other disorders in which the presence of MCP, includingMCP-1, MCP-2, and/or MCP-3, in a subject suffering from the disorder hasbeen shown to be or is suspected of being either responsible for thepathophysiology of the disorder or a factor that contributes to aworsening of the disorder. Accordingly, a disorder in which MCP activityis detrimental is a disorder in which inhibition of MCP activity isexpected to alleviate the symptoms and/or progression of the disorder.Such disorders may be evidenced, for example, by an increase in theconcentration of MCP in a biological fluid of a subject suffering fromthe disorder (e.g., an increase in the concentration of MCP-1 in serum,plasma, synovial fluid, etc. of the subject), which can be detected, forexample, using an anti-MCP antibody as described above. There arenumerous examples of disorders in which MCP activity is detrimental. Theuse of the antibodies and antibody portions of the invention in thetreatment of specific disorders is discussed further below.

The b-chemokines, particularly MCP-1, MCP-2 and MCP-3 have been shown toplay a role in pathological conditions associated with inflammation (VanCoillie et al. (1999) Cytokine & Growth Factor Rev. 10:61-86). MCP-1,MCP-2, and MCP-3 have all been shown to have potent chemotactic activityfor leukocytes, especially monocytes (van Coillie et al. (1999) Cytokine& Growth Factor Rev. 10:61-86). All three chemokines also share otherfunctions (e.g., glucosaminidase release, gelatinase B release, granzymeA) which combined with their chemotactic activity enable leukocytes tomigrate into tissues and towards sites of inflammation. Recruitment ofleukocytes to inflammatory sites is thought to contribute greatly to theinflammatory process. Inhibition of leukocyte recruitment via MCP-1antagonism (e.g., in MCP-1 knockout animals and in MCP-1 depletionstudies using anti-MCP-1 mAbs) has been shown to reduce leukocyteinfiltration (particularly monocyte recruitment) and is correlated withreduction in disease (van Coillie et al. (1999) Cytokine & Growth FactorRev. 10:61-86). Like MCP-1, MCP-2 and MCP-3 are also molecules withpotent chemotactic activity for monocytes, T lymphocytes, and basophils.Given their overlapping activities and the increased expression of allthree chemokines (MCP-1, MCP-2, and MCP-3) in human disease, blockade ofall three MCP molecules would be expected to have a greater beneficialeffect than just inhibition of MCP-1 alone.

Blockade of multiple MCP molecules (MCP-1, MCP-2 and MCP-3) would alsomore efficiently inhibit recruitment of certain cell types for whichMCP-1 is a poor chemotactic stimulus. Thus, while MCP-1 does notefficiently induce migration of eosinophils or resting neutrophils,MCP-2 is a potent chemotactic stimulus for eosinophils, and MCP-3 showsactivity against both eosinophils and neutrophils (van Coillie et al.(1999) Cytokine & Growth Factor Rev. 10:61-86). The antibodies andantibody fragments of the invention may be used to modulate the activityof these chemokines and affect the pathology of these disorders, andtherefore, may be used in therapeutic compositions for the treatment ofinflammatory conditions and pathological conditions associated withexpression of MCP molecules. In these embodiments, a subject isidentified as having one of the diseases to be treated, such as byexhibiting at least one sign or symptom of the disease or disorder. Atleast one antibody or antigen-binding fragment thereof of the inventionor compositions comprising at least one antibody or antigen-bindingfragment thereof of the invention is administered in a sufficient amountto alleviate at least one symptom of the disease or disorder, or toreduce the activity of at least one of MCP-1, MCP-2 or MCP-3.

1. Fibrotic Disease

In one embodiment of the invention, an antibody or antigen-bindingfragment thereof, having binding specificity for MCP-1, MCP-2 and/orMCP-3, e.g., an antibody or antigen-binding fragment comprising CDRsfrom either the 1A1 or 11K2 antibodies, is used in a method ofprevention or treatment of a subject suffering from a fibrotic disease.A “fibrotic disease” as used herein includes any condition marked by anincrease of interstitial fibrous tissue. MCPs are known to be associatedwith fibrotic conditions. For example, MCP-1 is a potent chemoattractantfor monocytes and has been implicated in a variety of inflammatory andfibrotic diseases, the pathogenesis of which is known to involveinfiltration and activation of monocytes (Zhang, et al (1994) J.Immunol. 153:4733-4741). Along with increased TGF-β and collagenproduction, fibrotic diseases are also characterized by increased levelsof MCP-1 (Antoniades et al. (1992) J. Immunol 89:5371-5375; Wada et al.(1996) FASEB J., 10:1418-1425; Saitoh et al. (1998) J. Clin. Lab. Anal.12:1-5; Hasegawa et al. (1999) Clin. Exp. Immunol. 117:159-165; Wada etal. (1999) Kidney Int. 65:995-1003; Wada et al. (2000) Kidney Int.58-1492-1499). Increased expression of MCP-1 during fibrotic diseaseshas been well characterized in both human and in rodent models. Inhumans, MCP-1 is up-regulated in idiopathic pulmonary fibrosis(Antoniades et al., supra), IgA nephropathy (Saitoh et al., supra),diabetic nephropathy (Wada et al. (2000), supra), lupus nephritis (Wadaet al., (1996), supra), crescentic glomerulonephritis (Wada, 1999),supra), and scleroderma (Hasegawa, supra). While not expressed in normaltissues, MCP-1 was highly expressed in the fibrotic skin and lungs ofscleroderma subjects, and the elevated levels of MCP-1 found in subjectserum correlated with the presence of fibrosis and with earlier onset ofscleroderma (Hasegawa, supra). MCP-1 expression also correlatedpositively with severity of renal fibrosis in diseases such as IgAnephropathy, diabetic nephropathy, lupus nephritis, and crescenticglomerulonephritis.

2. Oncogenic Disease

In another embodiment of the invention, an antibody or antigen-bindingfragment thereof, having binding specificity for MCP-1, MCP-2 and/orMCP-3, e.g., an antibody or antigen-binding fragment comprising CDRsfrom either the 1A1 or 11K2 antibodies, is used in a method ofprevention or treatment of a subject suffering from an oncogenic diseaseor cancer. MCPs are known to be associated with oncogenic conditions.For example, MCP-1 is a potent inducer of angiogenesis and plays animportant role in tumor growth. Evidence for a role of MCP-1 intumorigenesis involved treatment of immunodeficient mice bearing MCP-1producing human breast carcinoma cells with neutralizing anti-MCP-1 mAb(Salcedo, (2000) Blood 96:34-40). Treatment with anti-MCP-1 mAb resultedin significant increases in animal survival (mean survival increasedfrom 45 days to 75 days) and marked inhibition of tumor metastasis (60%decrease in lung metastatic index).

3. Immunopathologic Disease

In another embodiment of the invention, an antibody or antigen-bindingfragment thereof, having binding specificity for MCP-1, MCP-2 and/orMCP-3, e.g., an antibody or antigen-binding fragment comprising CDRsfrom either the 1A1 or 11K2 antibodies, is used in a method ofprevention or treatment of a subject suffering from an immunopathologicdisease. An “immunopathologic disease” as used herein is defined as anycondition associated with an immune response which is related to adisease. MCPs have been associated with immunopathologic conditions. Forexample, there is a strong link between MCP-1 expression andimmunopathologic disease in humans. Experiments usinggenetically-engineered mice and in vivo data using function-blockingantibodies to MCP-1 provide evidence supporting the validity of MCP-1antagonism in a variety of diseases characterized by mononuclearinfiltration. Included among these diseases is: atherosclerosis (MCP-1KO, CCR2 KO), arthritis (MCP-1 mAb), asthma (MCP-1 mAb),glomerulonephritis (MCP-1 KO, MCP-1 mAb), lupus nephritis (MCP-1 KO) andmultiple sclerosis (MCP-1 KO, MCP-1 mAb, CCR2 KO) (see, for example, Luet al. (1998) J. Exp. Med. 187601-608); Kurihara et al. (1997) J. Exp.Med. 186:1757-1762; Boring et al. (1997) J. Clin. Invest.100:2552-2561); Kuziel et al. (1997) PNAS 94:12053-12058; Blease et al.(2000) J. Immunol. 165:2603-2611; Traynor et al. (2000) J. Immunol.164:2021-2027; Boring et al. (1998) Nature 394:894-897; Dawson et al.(1999) Atherosclerosis 143:205-211; Fife et al. (2000) J. Exp. Med. 192:899-905; Izikson et al. (2000) J. Exp. Med. 192:1075-1080; Bird et al.(2000) Kidney Int. 57:129-136; MacLean et al., (2000) J. Immunol.165:165:6568-6575; Karpus et al. (1997) J. Leukoc. Biol. 62:681-687;Gonzalo et al. (1998) J. Exp. Med. 188:157-167). In all these cases,interference with the MCP-1 pathway resulted in dramatically reducedleukocyte infiltration, with monocyte recruitment being particularlyaffected. This dramatic reduction in monocyte recruitment correlatedwell with reduction in disease.

4. Other Disorders

In certain embodiments, the antibodies or antigen-binding fragments ofthe present invention are useful in the prevention or treatment ofglomerulonephritis, scleroderma, cirrhosis, multiple sclerosis, lupusnephritis, atherosclerosis, inflammatory bowel diseases or rheumatoidarthritis. In another embodiment, the antibodies or antigen-bindingfragments of the invention can be used to treat or prevent inflammatorydisorders, including, but not limited to, Alzheimer's, severe asthma,atopic dermatitis, cachexia, CHF-ischemia, coronary restinosis, Crohn'sdisease, diabetic nephropathy, lymphoma, psoriasis,fibrosis/radiation-induced, juvenile arthritis, stroke, inflammation ofthe brain or central nervous system caused by trauma, and ulcerativecolitis. Other inflammatory disorders which can be prevented or treatedwith the antibodies or antigen-binding fragments of the inventioninclude inflammation due to corneal transplantation, chronic obstructivepulmonary disease, hepatitis C, multiple myeloma, and osteoarthritis. Inanother embodiment, the antibodies or antigen-binding fragments of theinvention can be used to prevent or treat neoplasia, including, but notlimited to bladder cancer, breast cancer, head and neck cancer, kaposi'ssarcoma, melanoma, ovarian cancer, small cell lung cancer, stomachcancer, leukemia/lymphoma, and multiple myeloma. Additional neoplasiaconditions include, cervical cancer, colo-rectal cancer, endometrialcancer, kidney cancer, non-squamous cell lung cancer, and prostatecancer. In another embodiment, the antibodies or antigen-bindingfragments of the invention can be used to prevent or treat fibroticdisorders, including, but not limited to CHF-ischemia, coronaryrestenosis, diabetic vasculopathy, myocardial infarction/unstableangina, and radiation fibrosis. Additional examples of fibroticdisorders which can be treated in accordance with the invention includediabetic nephropathy, and impotence (Peyronie's). In another embodiment,the antibodies or antigen-binding fragments of the invention can be usedto prevent or treat neurodegenerative disorders, including, but notlimited to Alzheimer's, stroke, and traumatic brain or central nervoussystem injuries. Additional neurodegenerative disorders includeALS/motor neuron disease, diabetic peripheral neuropathy, diabeticretinopathy, Huntington's disease, macular degeneration, and Parkinson'sdisease.

In clinical applications, a subject is identified as having or at riskof developing a disease or disorder associated with detrimental MCPactivity, such as by exhibiting at least one sign or symptom of thedisease or disorder. At least one antibody or antigen-binding fragmentthereof of the invention or compositions comprising at least oneantibody or antigen-binding fragment thereof of the invention isadministered in a sufficient amount to treat at least one symptom of thedisease or disorder, or to reduce the activity of at least one of MCP-1,MCP-2 or MCP-3.

B. Animal Model for Testing Efficacy of Antibodies

Moreover, an antibody of the invention can be administered to anon-human mammal expressing a chemokine with which the antibodycross-reacts (e.g., a primate, pig or mouse) for veterinary purposes oras an animal model of human disease. Regarding the latter, such animalmodels may be useful for evaluating the therapeutic efficacy ofantibodies of the invention (e.g., testing of dosages and time coursesof administration). Examples of animal models which can be used forevaluating the therapeutic efficacy of antibodies or antigen-bindingfragments of the invention for preventing or treating glomerulonephritisinclude anti-induced glomerulonephritis (Wada et al. (1996) Kidney Int.49:761-767) and anti-thyl-induced glomerulonephritis (Schneider et al.(1999) Kidney Int. 56:135-144). Examples of animal models which can beused for evaluating the therapeutic efficacy of antibodies orantigen-binding fragments of the invention for preventing or treatingcolitis include a mouse model where colitis is TNBS-induced, asdescribed in Neurath et al. (1995) J Exp Med. 182(5):1281. Examples ofanimal models which can be used for evaluating the therapeutic efficacyof antibodies or antigen-binding fragments of the invention forpreventing or treating cirrhosis include carbon tetrachloride-inducedcirrhosis and liver fibrosis (Sakadamis et al. (2001) Res Exp Med200:137-54). Examples of animal models which can be used for evaluatingthe therapeutic efficacy of antibodies or antigen-binding fragments ofthe invention for preventing or treating multiple sclerosis includeexperimental autoimmune encephalomyelitis (EAE) (Link and Xiao (2001)Immunol. Rev. 184:117-128). Animal models can also be used forevaluating the therapeutic efficacy of antibodies or antigen-bindingfragments of the invention for preventing or treating lupus, for exampleusing the MRL-Fas^(lpr) mice (Schneider, supra; Tesch et al. (1999) J.Exp. Med. 190). Examples of animal models which can be used forevaluating the therapeutic efficacy of antibodies or antigen-bindingfragments of the invention for preventing or treating atherosclerosisinclude using mice deficient in apolipoprotein A, ApoE, and LDL R_(L)(Dansky et al. (1999) Arterioscler Thromb. Vasc. Biol. 19:1960-1968; Louet al., (1998) PNAS 95:12591-12595). Examples of animal models which canbe used for evaluating the therapeutic efficacy of antibodies orantigen-binding fragments of the invention for preventing or treatinginflammatory bowel disease (IBD) include TNBS-induced IBD, DSS-inducedIBD, and (Padol et al. (2000) Eur. J. Gastrolenterol. Hepatol 12:257;Murthy et al. (1993) Dig. Dis. Sci. 38:1722). Examples of animal modelswhich can be used for evaluating the therapeutic efficacy of antibodiesor antigen-binding fragments of the invention for preventing or treatingrheumatoid arthritis (RA) include adjuvant-induced RA, collagen-inducedRA, and collagen mAb-induced RA (Holmdahl et al., (2001) Immunol. Rev.184:184; Holmdahl et al., (2002) Ageing Res. Rev. 1:135; Van den Berg(2002) Curr. Rheumatol. Rep. 4:232).

In addition, animal models for evaluating the efficacy of antibodies orantigen-binding fragments of the invention in treating or preventinghuman fibrotic diseases, include rodent models of pulmonary (Brieland etal., (1992) Am. J. Respir. Cell. Mol. Biol. 7:134-139; Zhang et al.(1994) J. Immunol. 153:4733-4741; Johnston et al. (1998) Exp. Lung Res.24:321-337), vascular (Furukawa et al. (1999) Circ. Res. 84:306-314),and renal (Lloyd et al. (1997) J. Exp. Med. 185:1371-1380; Fujinaka etal. (1997) J. Am. Soc. Nephrol. 8:1174-1178; Schneider, supra; Tesch etal. (1999) J. Exp. Med. 190: 1813-1824; Tesch et al. (1999) J. Clin.Invest. 103:73-80) fibrosis. Alport's model of renal fibrosis can alsobe used to evaluate the efficacy of the antibodies or antigen-bindingfragments of the invention.

C. Treatment Regimes and Dosages

In prophylactic applications, pharmaceutical compositions or medicamentsare administered to a subject suffering from a disorder in which MCPactivity is detrimental, in an amount sufficient to eliminate or reducethe risk, lessen the severity, or delay the outset of the disorder,including biochemical, histologic and/or behavioral symptoms of thedisorder, its complications and intermediate pathological phenotypespresenting during development of the disorder. In therapeuticapplications, compositions or medicants are administered to a subjectsuspected of, or already suffering from such a disorder in an amountsufficient to cure, or at least partially arrest, the symptoms of thedisorder (biochemical, histologic and/or behavioral), including itscomplications and intermediate pathological phenotypes in development ofthe disorder.

In some methods, administration of agent reduces or eliminatesinflammation associated with MCPs. An amount adequate to accomplishtherapeutic or prophylactic treatment is defined as a therapeutically-or prophylactically-effective dose. In both prophylactic- andtherapeutic regimes, agents are usually administered in several dosagesuntil a sufficient immune response has been achieved. The term “immuneresponse” or “immunological response” includes the development of ahumoral (antibody mediated) and/or a cellular (mediated byantigen-specific T cells or their secretion products) response directedagainst an antigen in a recipient subject. Such a response can be anactive response, i.e., induced by administration of immunogen, or apassive response, i.e., induced by administration of immunoglobulin orantibody or primed T-cells.

An “immunogenic agent” or “immunogen” is capable of inducing animmunological response against itself on administration to a mammal,optionally in conjunction with an adjuvant. Typically, the immuneresponse is monitored and repeated dosages are given if the immuneresponse starts to wane.

Effective doses of the compositions of the present invention, for thetreatment of the above described conditions vary depending upon manydifferent factors, including means of administration, target site,physiological state of the subject, whether the subject is human or ananimal, other medications administered, and whether treatment isprophylactic or therapeutic. Usually, the subject is a human butnon-human mammals including transgenic mammals can also be treated.Treatment dosages need to be titrated to optimize safety and efficacy.

For passive immunization with an antibody, the dosage ranges from about0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the host bodyweight. For example dosages can be 1 mg/kg body weight or 10 mg/kg bodyweight or within the range of 1-10 mg/kg, preferably at least 1 mg/kg.Subjects can be administered such doses daily, on alternative days,weekly or according to any other schedule determined by empiricalanalysis. An exemplary treatment entails administration in multipledosages over a prolonged period, for example, of at least six months.Additional exemplary treatment regimes entail administration once perevery two weeks or once a month or once every 3 to 6 months. Exemplarydosage schedules include 1-10 mg/kg or 15 mg/kg on consecutive days, 30mg/kg on alternate days or 60 mg/kg weekly. In some methods, two or moremonoclonal antibodies with different binding specificities areadministered simultaneously, in which case the dosage of each antibodyadministered falls within the ranges indicated.

Antibody is usually administered on multiple occasions. Intervalsbetween single dosages can be weekly, monthly or yearly. Intervals canalso be irregular as indicated by measuring blood levels of antibody toMCPs in the subject. In some methods, dosage is adjusted to achieve aplasma antibody concentration of 1-1000 μg/ml and in some methods 25-300μg/ml. Alternatively, antibody can be administered as a sustainedrelease formulation, in which case less frequent administration isrequired. Dosage and frequency vary depending on the half-life of theantibody in the subject. In general, human antibodies show the longesthalf-life, followed by humanized antibodies, chimeric antibodies, andnonhuman antibodies.

The dosage and frequency of administration can vary depending on whetherthe treatment is prophylactic or therapeutic. In prophylacticapplications, compositions containing the present antibodies or acocktail thereof are administered to a subject not already in thedisease state to enhance the subject's resistance. Such an amount isdefined to be a “prophylactic effective dose.” In this use, the preciseamounts again depend upon the subject's state of health and generalimmunity, but generally range from 0.1 to 25 mg per dose, especially 0.5to 2.5 mg per dose. A relatively low dosage is administered atrelatively infrequent intervals over a long period of time. Somesubjects continue to receive treatment for the rest of their lives.

In therapeutic applications, a relatively high dosage (e.g., from about1 to 200 mg of antibody per dose, with dosages of from 5 to 25 mg beingmore commonly used) at relatively short intervals is sometimes requireduntil progression of the disease is reduced or terminated, andpreferably until the subject shows partial or complete amelioration ofsymptoms of disease. Thereafter, the patent can be administered aprophylactic regime.

Doses for nucleic acids encoding antibodies range from about 10 ng to 1g, 100 ng to 100 mg, 1 μg to 10 mg, or 30-300 μg DNA per subject. Dosesfor infectious viral vectors vary from 10-100, or more, virions perdose.

Therapeutic agents can be administered by parenteral, topical,intravenous, oral, subcutaneous, intraarterial, intracranial,intraperitoneal, intranasal or intramuscular means for prophylacticand/or therapeutic treatment. The most typical route of administrationof an immunogenic agent is subcutaneous although other routes can beequally effective. The next most common route is intramuscularinjection. This type of injection is most typically performed in the armor leg muscles. In some methods, agents are injected directly into aparticular tissue where deposits have accumulated, for exampleintracranial injection. Intramuscular injection or intravenous infusionare preferred for administration of antibody. In some methods,particular therapeutic antibodies are injected directly into thecranium. In some methods, antibodies are administered as a sustainedrelease composition or device, such as a Medipad™ device.

Agents of the invention can optionally be administered in combinationwith other agents that are at least partly effective in treatment ofMCP-associated disorders.

D. Pharmaceutical Compositions

The therapeutic compositions of the invention include at least oneantibody or antibody fragment of the invention in a pharmaceuticallyacceptable carrier. A “pharmaceutically acceptable carrier” refers to atleast one component of a pharmaceutical preparation that is normallyused for administration of active ingredients. As such, a carrier maycontain any pharmaceutical excipient used in the art and any form ofvehicle for administration. The compositions may be, for example,injectable solutions, aqueous suspensions or solutions, non-aqueoussuspensions or solutions, solid and liquid oral formulations, salves,gels, ointments, intradermal patches, creams, lotions, tablets,capsules, sustained release formulations, and the like. Additionalexcipients may include, for example, colorants, taste-masking agents,solubility aids, suspension agents, compressing agents, entericcoatings, sustained release aids, and the like.

Agents of the invention are often administered as pharmaceuticalcompositions comprising an active therapeutic agent, and a variety ofother pharmaceutically acceptable components. See Remington'sPharmaceutical Science (15th ed., Mack Publishing Company, Easton, Pa.(1980)). The preferred form depends on the intended mode ofadministration and therapeutic application. The compositions can alsoinclude, depending on the formulation desired,pharmaceutically-acceptable, non-toxic carriers or diluents, which aredefined as vehicles commonly used to formulate pharmaceuticalcompositions for animal or human administration. The diluent is selectedso as not to affect the biological activity of the combination. Examplesof such diluents are distilled water, physiological phosphate-bufferedsaline, Ringer's solutions, dextrose solution, and Hank's solution. Inaddition, the pharmaceutical composition or formulation may also includeother carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenicstabilizers and the like.

Pharmaceutical compositions can also include large, slowly metabolizedmacromolecules such as proteins, polysaccharides such as chitosan,polylactic acids, polyglycolic acids and copolymers (such as latexfunctionalized Sepharose™, agarose, cellulose, and the like), polymericamino acids, amino acid copolymers, and lipid aggregates (such as oildroplets or liposomes). Additionally, these carriers can function asimmunostimulating agents (i.e., adjuvants).

For parenteral administration, agents of the invention can beadministered as injectable dosages of a solution or suspension of thesubstance in a physiologically acceptable diluent with a pharmaceuticalcarrier that can be a sterile liquid such as water oils, saline,glycerol, or ethanol. Additionally, auxiliary substances, such aswetting or emulsifying agents, surfactants, pH buffering substances andthe like can be present in compositions. Other components ofpharmaceutical compositions are those of petroleum, animal, vegetable,or synthetic origin, for example, peanut oil, soybean oil, and mineraloil. In general, glycols such as propylene glycol or polyethylene glycolare preferred liquid carriers, particularly for injectable solutions.Antibodies can be administered in the form of a depot injection orimplant preparation, which can be formulated in such a manner as topermit a sustained release of the active ingredient. An exemplarycomposition comprises monoclonal antibody at 5 mg/mL, formulated inaqueous buffer consisting of 50 mM L-histidine, 150 mM NaCl, adjusted topH 6.0 with HCl.

Typically, compositions are prepared as injectables, either as liquidsolutions or suspensions; solid forms suitable for solution in, orsuspension in, liquid vehicles prior to injection can also be prepared.The preparation also can be emulsified or encapsulated in liposomes ormicro particles such as polylactide, polyglycolide, or copolymer forenhanced adjuvant effect, as discussed above (see Langer, Science 249:1527 (1990) and Hanes, Advanced Drug Delivery Reviews 28:97 (1997)). Theagents of this invention can be administered in the form of a depotinjection or implant preparation, which can be formulated in such amanner as to permit a sustained or pulsatile release of the activeingredient.

Additional formulations suitable for other modes of administrationinclude oral, intranasal, and pulmonary formulations, suppositories, andtransdermal applications. For suppositories, binders and carriersinclude, for example, polyalkylene glycols or triglycerides; suchsuppositories can be formed from mixtures containing the activeingredient in the range of 0.5% to 10%, preferably 1%-2%. Oralformulations include excipients, such as pharmaceutical grades ofmannitol, lactose, starch, magnesium stearate, sodium saccharine,cellulose, and magnesium carbonate. These compositions take the form ofsolutions, suspensions, tablets, pills, capsules, sustained releaseformulations or powders and contain 10%-95% of active ingredient,preferably 25%-70%.

Topical application can result in transdermal or intradermal delivery.Topical administration can be facilitated by co-administration of theagent with cholera toxin or detoxified derivatives or subunits thereofor other similar bacterial toxins (See Glenn et al., Nature 391, 851(1998)). Co-administration can be achieved by using the components as amixture or as linked molecules obtained by chemical crosslinking orexpression as a fusion protein.

Alternatively, transdermal delivery can be achieved using a skin path orusing transferosomes (Paul et al., Eur. J. Immunol. 25:3521 (1995); Cevcet al., Biochem. Biophys. Acta 1368:201-15 (1998)).

III. Monitoring the Course of Treatment

The invention provides methods of monitoring treatment in a subjectsuffering from a disorder in which MCP activity is detrimental, i.e.,for monitoring a course of treatment being administered to a subject.The methods can be used to monitor both therapeutic treatment onsymptomatic subjects and prophylactic treatment on asymptomaticsubjects. In particular, the methods are useful for monitoring passiveimmunization (e.g., measuring level of administered antibody).

Some methods entail determining a baseline value, for example, of anantibody level or profile in a subject, before administering a dosage ofagent, and comparing this with a value for the profile or level aftertreatment. A significant increase (i.e., greater than the typical marginof experimental error in repeat measurements of the same sample,expressed as one standard deviation from the mean of such measurements)in value of the level or profile signals a positive treatment outcome(i.e., that administration of the agent has achieved a desiredresponse). If the value for immune response does not changesignificantly, or decreases, a negative treatment outcome is indicated.

In other methods, a control value (i.e., a mean and standard deviation)of level or profile is determined for a control population. Typicallythe individuals in the control population have not received priortreatment. Measured values of the level or profile in a subject afteradministering a therapeutic agent are then compared with the controlvalue. A significant increase relative to the control value (e.g.,greater than one standard deviation from the mean) signals a positive orsufficient treatment outcome. A lack of significant increase or adecrease signals a negative or insufficient treatment outcome.Administration of agent is generally continued while the level isincreasing relative to the control value. As before, attainment of aplateau relative to control values is an indicator that theadministration of treatment can be discontinued or reduced in dosageand/or frequency.

In other methods, a control value of the level or profile (e.g., a meanand standard deviation) is determined from a control population ofindividuals who have undergone treatment with a therapeutic agent andwhose levels or profiles have plateaued in response to treatment.Measured values of levels or profiles in a subject are compared with thecontrol value. If the measured level in a subject is not significantlydifferent (e.g., more than one standard deviation) from the controlvalue, treatment can be discontinued. If the level in a subject issignificantly below the control value, continued administration of agentis warranted. If the level in the subject persists below the controlvalue, then a change in treatment may be indicated.

In other methods, a subject who is not presently receiving treatment buthas undergone a previous course of treatment is monitored for antibodylevels or profiles to determine whether a resumption of treatment isrequired. The measured level or profile in the subject can be comparedwith a value previously achieved in the subject after a previous courseof treatment. A significant decrease relative to the previousmeasurement (i.e., greater than a typical margin of error in repeatmeasurements of the same sample) is an indication that treatment can beresumed. Alternatively, the value measured in a subject can be comparedwith a control value (mean plus standard deviation) determined in apopulation of subjects after undergoing a course of treatment.Alternatively, the measured value in a subject can be compared with acontrol value in populations of prophylactically treated subjects whoremain free of symptoms of disease, or populations of therapeuticallytreated subjects who show amelioration of disease characteristics. Inall of these cases, a significant decrease relative to the control level(i.e., more than a standard deviation) is an indicator that treatmentshould be resumed in a subject.

The tissue sample for analysis is typically blood, plasma, serum, mucousfluid or cerebrospinal fluid from the subject. The sample is analyzed,for example, for levels or profiles of antibodies to Aβ peptide, e.g.,levels or profiles of humanized antibodies. ELISA methods of detectingantibodies specific to MCPs are described in the Examples section.

The antibody profile following passive immunization typically shows animmediate peak in antibody concentration followed by an exponentialdecay. Without a further dosage, the decay approaches pretreatmentlevels within a period of days to months depending on the half-life ofthe antibody administered. For example the half-life of some humanantibodies is of the order of 20 days.

In some methods, a baseline measurement of antibody to MCPs in thesubject is made before administration, a second measurement is made soonthereafter to determine the peak antibody level, and one or more furthermeasurements are made at intervals to monitor decay of antibody levels.When the level of antibody has declined to baseline or a predeterminedpercentage of the peak less baseline (e.g., 50%, 25% or 10%),administration of a further dosage of antibody is administered. In somemethods, peak or subsequent measured levels less background are comparedwith reference levels previously determined to constitute a beneficialprophylactic or therapeutic treatment regime in other subjects. If themeasured antibody level is significantly less than a reference level(e.g., less than the mean minus one standard deviation of the referencevalue in population of subjects benefiting from treatment)administration of an additional dosage of antibody is indicated.

Additional methods include monitoring, over the course of treatment, anyart-recognized physiologic symptom (e.g., physical or mental symptom)routinely relied on by researchers or physicians to diagnose or monitordisorders associated with detrimental MCP activity.

The invention further provides kits for performing the monitoringmethods described above. Typically, such kits contain an agent thatspecifically binds to antibodies to MCPs, including MCP-1, MCP-2, and/orMCP-3. The kit can also include a label. For detection of antibodies toMCPs, the label is typically in the form of labeled anti-idiotypicantibodies. For detection of antibodies, the agent can be suppliedprebound to a solid phase, such as to the wells of a microliter dish.Kits also typically contain labeling providing directions for use of thekit. The labeling may also include a chart or other correspondenceregime correlating levels of label with levels of antibodies to MCPs.The term labeling refers to any written or recorded material that isattached to, or otherwise accompanies a kit at any time during itsmanufacture, transport, sale or use. For example, the term labelingencompasses advertising leaflets and brochures, packaging materials,instructions, audio or videocassettes, computer discs, as well aswriting imprinted directly on kits.

The invention also provides diagnostic kits, for example, research,detection and/or diagnostic kits (e.g., for performing in vivo imaging).Such kits typically contain an antibody for binding to an epitope of anMCP. Preferably, the antibody is labeled or a secondary labeling reagentis included in the kit. Preferably, the kit is labeled with instructionsfor performing the intended application, for example, for performing anin vivo imaging assay. Exemplary antibodies are those described herein.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication, as well as the figures and the Sequence Listing, areincorporated herein by reference.

EXAMPLES I. Characterization of Anti-Chemokine Monoclonal AntibodySupernatants Example 1 ELISA Screening

MaxiSorp 384 well plates were coated with 15-20 μl antigen in PBS.Recombinant purified human antigens included: MCP-1, MCP-2, MCP-3,MCP-4, IL-8, eotaxin, fractalkine, Gcp-2, DC-CK (Gcp-2 and DC-CK arealso chemokines). Plates were incubated with antigen for 2 hours at 37°C. or overnight at 4° C. Non-specific sites were blocked with 80 μl/wellof 1% BSA/PBS for 1 hour at room temperature. Plates were washed and 15μl of hybridoma supernatant was added to each well and incubated for 1hour at room temperature. Plates were washed and wells were incubatedwith 20 μl/well of a 1:25 000 dilution of goat anti-mouse IgG peroxidaseconjugate (Jackson Catalog Number 515-036-003). Plates were incubatedfor 1 hour at room temperature, washed, and 20 μl/well of substrate(TMB, tetramethylbenzidine, Jackson, Catalog Number 515-036-062) wasadded. Reaction was allowed to proceed and stopped by addition of 20μl/well of 2M H₂SO₄. Reactive clones were picked for further analysis.Isotyping of hybridoma supernatants was performed by antigen-dependentELISA. Briefly, wells were coated with 50 μl of human MCP-1 (5 μg/ml)for 1 hour at 37° C. Wells were washed 4 times and blocked with PBS/1%BSA. Isotyping of hybridoma supernatants was then performed using amouse immunoglobulin screening/isotyping kit (Zymed Laboratories, SanFrancisco, Calif.) as recommended by the manufacturer. Specificities ofthe antibodies and clones obtained are shown in Table 1.

TABLE 3 Panel of MCP mAbs Specificity Block MCP-4/IL-8/ MCP-1Eotaxin/Frac Affinity Sub- ligand Fusion Immunogen Clone MCP-1 MCP-2MCP-3 Gcp-2/DC-CK (Biacore) Type binding IA1 MCP-1 1M-11 ++++ − − − ++++IgG1 ++++ 3N10 ++++ − − − ++++ IgG1 ++++ IA7 MCP-1 11K2 ++++ ++++ ++++ −++++ IgG1 ++++ MCP-2 7F7 ++++ − − − ++++ IgG1 +++ MCP-3 6D21 ++++ ++++++ − ++++ IgG1 ++++ 6E11 ++ − + − +++ IgM +++ 1A1 +++ + + − ++++ IgG1++++ IA8 MCP-1 4N4 ++++ ++ ++ − ++++ IgG1 ++++ MCP-2 5A13 +++ ++ ++ −+++ IgG1 ++++ IA9 MCP-1 5J23 ++++ ++ − − ++++ IgG1 ++++ MCP-2 6I5 +++++++ +++ − ++++ IgG1 ++++ MCP-3 7H1 ++++ ++++ ++++ − ++++ IgG1 ++++ IA10MCP-1 4N9 + − ++ − ++++ IgG1 +++ MCP-2 2O24 ++ − ++ − ++++ IgG1 +++MCP-3 9H23 ++ − + − +++ IgG1 +++ 9B11 ++++ − ++++ − ++++ IgG1 +++ 9B12 +− +++ − ++++ IgG1 +++ 9C11 ++++ − ++++ − ++++ IgG1 +++ 10D18 ++++ − ++++− ++++ IgG1 ++++ 12F15 +++ − +++ − ++++ IgG1 ++++ MCP-1 D9 ++++ − − −(MCP-4, IL-8 +++ and eotaxin only tested) + and − indicate the relativeamount of binding of the antibody to the various immobilized ligands.

In addition, both 1A1 and 11K2 mAbs recognize primate MCP-1. Plates werecoated with 1 μg/ml of chemically synthesized chemokines (correspondingto the cynomolgus and rhesus MCP-1 sequences) and probed with 10 μg/mlof monoclonal antibodies, including MOPC21 (IgG1b control antisera),11K2, 3N10, 1A1, D9, and 1M11, as described above. Results demonstratethat all of the above mAbs, including 1A1 and 11K2, and with theexception of the isotype control mAb MOPC21, also recognize primateMCP-1.

Example 2 Binding Assay

¹²⁵I labeled MCP-1 (2200 Ci/mmol) was purchased from NEN Life Sciences(Boston, Mass.). Hybridoma supernatants (50 μl) were pre-incubated with1 nM ¹²⁵I MCP-1 (50 μl) for 60 minutes at room temperature prior to theaddition of the CCR2-expressing human monocyte cell line, THP-1. THP-1cells (1×10⁷ cells/ml; 50 μl) were resuspended in binding buffer (50 mMHepes, 1 mM CaCl₂, 5 mM MgCl₂, 0.5% BSA) and added to the combination of¹²⁵I labeled MCP-1 and hybridoma supernatant, and incubated at 4° C. for60 minutes. Cells were then washed 3 times by centrifugation in washbuffer (50 mM Hepes, 1 mM CaCl₂, 5 mM MgCl₂, 500 mM NaCl and 0.5% BSA).Amount of bound ¹²⁵I labeled MCP-1 was then quantitated for γ-emission.Pre-incubation of THP-1 cells (1×10⁷ cells/ml; 50 μl) with unlabeledMCP-1 (500 nM; 50 μl) for 60 minutes at 4° C. prior to addition of ¹²⁵Ilabeled MCP-1 (1 nM; 50 μl) served as a negative control. The positivecontrol represents binding of ¹²⁵I labeled MCP-1 to THP-1 cells in theabsence of MCP-1 hybridoma supernatant. The results are shown in Table2.

TABLE 4 Data of block ligand ([I¹²⁵] MCP-1) binding assay from γ CounterCPM CPM Fusion Immunogen Clone # (30/10/00) (22/11/00) IA1 MCP-1 1M-11499 456 45 40 3N10 379 495 50 46 IA7 MCP-1 11K2 103 148 50 49 MCP-2 7F7394 199 189 197 MCP-3 6D21 145 108 47 42 6E11 850 894 378 323 1A1 194772 47 52 IA8 MCP-1 4N4 40 47 MCP-2 5A13 50 44 IA9 MCP-1 5J23 478 280 5947 MCP-2 6I5 677 678 44 31 MCP-3 7H1 53 207 59 77 IA10 MCP-1 4N9 776 987306 340 MCP-2 2O24 936 869 226 357 MCP-3 9H23 293 226 9B11 679 892 238201 9B12 834 657 304 265 9C11 605 512 241 252 10D18 485 444 174 31012F15 421 344 281 213 12K14 406 918 Negative control 836 461 68 143Positive control 5861 3447 2084 2933

Example 3 Inhibition of Chemotaxis in Response to MCP-1

A 5 μm pore size ChemoTX plate (Neuroprobe) was used to assess thechemotactic response of THP-1 human monocytic cells. Hybridomasupernatants containing MCP-1 at 10 ng/ml, or RPMI with 10% FBS with orwithout 10 ng/ml chemokine, was added to the lower chamber of the plate.THP-1 cells at 2×106 cells/ml were layered on top. The plate wasincubated for 2 hours at 37° C. in 5% CO2. The filter was removed andthe number of cells that migrated into the lower chamber was determinedusing Promega Cell Titer reagent. The number of cells was Calculatedusing a standard curve (n=4, error bars=standard deviation). The resultsdemonstrated that antibodies 11K2, 7F7, 6D21 and 7H1 were all able toinhibit MCP-1-induced chemotaxis, although 11K2 and 6D21 were the mosteffective.

II. Characterization of Purified Anti-Chemokine Monoclonal AntibodiesExample 4 Chemokine Specificity and Binding Assays

ELISA specificity assays were performed using purified monoclonalantibodies to confirm the binding specificities of the supernatantMCP-specific monoclonal antibodies described above. Antibodies werepurified by Protein A affinity column chromatography, according tostandard methods known in the art.

ELISA was performed as previously described in Example 1. Briefly,MaxiSorp 384 well plates were coated with 15-20 μl antigen in PBS.Recombinant purified human antigens included: MCP-1, MCP-2, MCP-3,MCP-4, IL-8, eotaxin, murine MCP-1 (JE), Murine MCP-3, murine MCP-5, andrat MCP-1. All antigens, including MCP-3, were immobilized. Plates werewashed and purified monoclonal antibody (10 μg/ml) was added to eachwell and incubated for 1 hour at room temperature. Plates were washedand wells were incubated with 20 μl/well of a 1:25,000 dilution of goatanti-mouse IgG peroxidase conjugate (Jackson Catalog Number515-036-003). Plates were incubated for 1 hour at room temperature,washed, and 20 μl/well of substrate (TMB, tetramethylbenzidine, Jackson,Catalog Number 515-036-062) was added. Reaction was allowed to proceedand stopped by addition of 20 μl/well of 2M H₂SO₄. Specificities of thepurified antibodies and are shown in Table 3. Antibodies 1A1 boundspecifically to hMCP-1, hMCP-2, hMCP-3, and mMCP-1. Antibodies 11K2,4N4, 5A13, 6D21, 6I5, and 7H1 bound specifically to hMCP-1, hMCP-2,hMCP-3, mMCP-1, mMCP-3, and mMCP-5.

TABLE 5 ELISA performed using purified MCP mAbs. mMCP-1 hMCP-1 hMCP-2hMCP-3 hMCP-4 (JE) mMCP-3 mMCP-5 rtMCP-1 hIL8 hEotaxin 1A1 + + + − + − −− − − 4N4 + + + − + + + − − − 5A13 + + + − + + + − − − 6D21 + + +− + + + − − − 6I5 + + + − + + + − − − 7H1 + + + − + + + − − − 11K2 + + +− + + + − − − D9 + − − − − − − − − − 1M11 + − − − − ND ND ND ND ND3N10 + − − − − ND ND ND ND ND 2O24 + − + − − ND ND ND ND ND 9B11 + − + −− ND ND ND ND ND 9B12 + − + − − ND ND ND ND ND 9C11 + − + − − ND ND NDND ND 5J23 + + +/− − + ND ND − − −

Binding assays were performed using purified monoclonal antibodies toconfirm results obtained with the supernatants. Binding assays wereperformed as described in Example 2. Briefly, ¹²⁵I labeled MCP-1 (2200Ci/mol) was purchased from NEN Life Sciences (Boston, Mass.). Purifiedmonoclonal antibodies at various concentrations (33 nM, 3.3 nM and 0.33nM) were pre-incubated with 1 nM ¹²⁵I MCP-1 (50 μl) for 60 minutes atroom temperature prior to addition of the CCR2-expressing human monocytecell line, THP-1. THP-1 cells (1×10⁷ cells/ml; 50 μl) were resuspendedin binding buffer (50 mM Hepes, 1 mM CaCl₂, 5 mM MgCl₂, 0.5% BSA) andadded to the combination of ¹²⁵I labeled MCP-1 and purified mAb, andincubated at 4° C. for 60 minutes. Cells were then washed 3 times bycentrifugation in wash buffer (50 mM Hepes, 1 mM CaCl₂, 5 mM MgCl₂, 500mM NaCl, and 0.5% BSA). Amount of bound ¹²⁵I labeled MCP-1 was thenquantitated for γ-emission. Results from the binding assays are shown inTable 4. This study demonstrates that many of the studied monoclonalantibodies, including 1A1 and 11K2, were effective at blocking hMCP-1binding.

TABLE 6 Purified mAb binding assay Block hMCP-1 cell Antibody binding1A1 + 4N4 + 5A13 + 6D21 + 6I5 + 7H1 + 11K2 + D9 + 1M11 + 3N10 + 2O24 −9B11 − 9B12 − 9C11 − 5J23 +

Example 5 Inhibition of Monocyte Chemotaxis by Anti-Chemokine MonoclonalAntibodies A. MCP-1 and MCP-2 Chemotaxis Assay

A 5 μm pore size ChemoTX plate (Neuroprobe) was used to assess thechemotactic response of THP-1 human monocytic cells. Purified monoclonalantibodies (100 μg/ml) 11K2, 1A1, D9, and 2O24 were added in combinationwith and without MCP-1 (23 nM), MCP-2 (56 nM), and MCP-1/MCP-2 (2.3 nMMCP-1 and 56 nM MCP-2), to the lower chamber of the plate. THP-1 cellsat 2×10⁶ cells/ml were layered on top. The plate was incubated for 4hours at 37° C. in 5% CO₂. The filter was removed and the number ofcells that migrated into the lower chamber was determined using PromegaCell Titer reagent.

The results show that pan-monoclonal antibodies 11K2 and 1A1 wereeffective at inhibiting chemotaxis in the presence of both MCP-1 andMCP-2 (FIG. 1). The results also demonstrate that antibodies D9 and 2O24can inhibit chemotaxis which is induced by MCP-1 alone. Furthermore, asshown in FIG. 2, antibodies 1M11 and 3N10 can inhibit THP-1 chemotaxisinduced by human MCP-1, and antibody 5J23 can inhibit chemotaxis inducedby human and mouse MCP-1. In sum, chemotaxis to the combination of MCP-1and MCP-2 is inhibited by 11K2 and 1A1, but is not observed byantibodies D9 and 2O24, which are MCP-1-specific mAb (D9 and 2O24).

Within the pool of monoclonal antibodies studied, there are three groupswhich arise based on their ability to recognize certain MCP antigens.Monoclonal antibodies 1A1 and 11K2 recognize MCP-1, MCP-2 andimmobilized MCP-3. Monoclonal antibodies 1M11 and 3N10 recognize MCP-1,and antibody 2O24 recognizes MCP-1 and MCP-3. Antibody 5J23 recognizesmouse MCP-1 and recognizes only human MCP-1 and human MCP-2.

Results from a separate experiment using the ChemoTX plate (Neuroprobe)assay are shown below in Table 5. The protocol for this experiment wasthe same as previously described, except a titration of mAb was used incombination with fixed MCP concentrations (concentrations of MCPs areshown below in Table 5). The results described in Table 5 demonstratethat mAbs 11K2 and 1A1 are effective at inhibiting huMCP-1, huMCP-2,muMCP-1, and muMCP-5-induced chemotaxis.

TABLE 7 11K2 and 1A1 inhibit THP-1 chemotaxis towards human MCP-1, humanMCP-2, mouse MCP-1 and mouse MCP-5 Human ND₅₀ (nM) MCP-1(2.3 nM)MCP-2(56 nM) MCP-3(11.8 nM) MCP-4(58 nM) Commercial 10.0 33.0  2.6 11.51A1 1.4 47.5 No Inhib No Inhib 11K2 1.3 52.0 No Inhib No Inhib D9 5.8 NoInhib No Inhib No Inhib 2O24 1000.0 No Inhib 143.5 No Inhib Murine ND₅₀(nM) MCP-1(1.4 nM) MCP-2 MCP-3(59 nM) MCP-4 MCP-5(0.54 nM) Commercial3.2 ND 52.0 ND  0.1 1A1 1.7 ND No Inhib ND 13.5 11K2 2.1 ND No Inhib ND19.5 D9 No Inhib ND No Inhib ND No Inhib 2O24 No Inhib ND No Inhib ND NoInhib No Inhib = less than 50% Neutralization at 3 uMB. Inhibition of Chemotaxis by Cytokines Secreted from RA Fibroblasts

Prior to studying the ability of purified monoclonal antibodies 1A1,11K2, D9, 2O24, and 5D3-F7 (BD Biosciences, Pharmingen, San Diego,Calif.), to inhibit chemotaxis from chemokines secreted from stimulatedRA fibroblasts, a study of the different types of chemokines secreted byRA (rheumatoid arthritis) fibroblasts in response to inflammatorychemokines was performed. RA fibroblasts were exposed for 48 hours to500 U/ml IFN-γ, IFN-γ and 10 ng/ml of IL1β, or media (as a control).Results showed that IFN-γ alone induced low levels of MCP-1, MCP-2,MCP-3, and very low levels of IP10. IFN-γ exposure alone did not induceexpression of Rantes, IL-8, Mip1α, or Mip1β. In contrast, thecombination of IFN-γ and 10 ng/ml of IL1β induced about 27 ng/ml ofMCP-1, 31 ng/ml of MCP-2, 9 ng/ml of MCP-3, and 55 ng/ml of IL-8. Thecombination of IFN-γ and 10 ng/ml of IL1β also yielded low levels ofRantes, IP10, and Mip1α. The media alone control did not induce anychemokine secretion.

The ability of purified monoclonal antibodies to inhibit monocytechemotaxis to cytokines secreted from these stimulated RA fibroblastswas then studied. Supernatant from RA fibroblasts which were exposed toeither media alone, IFN-γ alone, or the combination of IFN-γ and IL-1β,were each tested for their chemotactic ability using human THP-1 cells.As a control, supernatant from unstimulated RA fibroblasts into whichIFN-γ (500 U/ml) and IL1β (10 ng/ml) was spiked was used. Thissupernatant (spike) control was used to evaluate the direct effects ofIL1β and IFN-γ on chemotaxis. As shown in FIG. 3, monocyte chemotaxismediated by cytokines secreted from stimulated RA fibroblasts wasinhibited by MCP mAbs 1A1 and 11K2. MCP-1-specific antibody D9 was alsoeffective at inhibiting chemotaxis in all experimental groups.

Example 6 MCP-1-Induced Calcium Flux Assay for Monoclonal 11K2

The MCP-1-induced calcium flux assay was performed according to standardprocedure. Briefly, monoclonal antibody 11K2 and a chemokine (MCP-1 orMCP-2) were mixed at 200× concentration and pre-incubated for one hour.This mixture was then added to THP-1 cells stirring in a cuvette in afluorimeter at t=30 sec. Calcium flux was measured by a change influorescence of Indo-1. Results show that MCP-1-induced calcium flux inTHP-1 cells was blocked by 11K2 (FIG. 4).

Example 7 Agonist Effect at Low Antibody Concentrations of 11K2 and 1A1A. Chemotaxis Assay

Chemotaxis assays were performed as previously described withrecombinant MCP-2 and using low concentrations of monoclonal antibodies11K2 and 1A1. The results from the chemotaxis assay showed that at a lowconcentration, monoclonal antibodies 11K2 and 1A1 increased MCP-2mediated chemotaxis. As shown in FIG. 5A, there was an increase inchemotaxis observed with low antibody concentrations (ranging from about1-15 nM) of 11K2 and 1A1, in contrast to the MCP-2 mAb 281 (RD Systems,Minneapolis, Minn.). The agonist effect was not seen with the Fabfragment of 11K2 or 1A1, and was MCP-2-specific. As shown in FIG. 5B,low concentrations of the 11K2 Fab fragment did not result in achemotactic increase in response to MCP-2 and showed only antagonistactivity.

B. Calcium Flux Assay

A calcium flux assay was performed as previously described, except a lowconcentration of 11K2 monoclonal antibody (16.5 nM) was also included.The results (FIG. 6) demonstrate that low concentrations of 11K2exposure results in agonistic activity in a MCP-2 calcium flux assay. Inaddition, however, 11K2 Fab and F(ab)₂ fragments are inhibitory in thesame assay (FIG. 6).

Example 8 Binding Affinity Measurement of Monoclonal 11K2 and 1A1

To measure the affinity of MCP mAb and Fab molecules for soluble MCPmolecules, a kinetic exclusion assay was utilized and affinity measuredusing a KinExA instrument (Sapidyne Instruments Inc., Boise, Id.).

Polymethylmethacrylate beads activated with NHS were coated with 10 μgrecombinant human MCP-1 in 1 ml buffer. The beads were packed into acolumn in the KinExA instrument for each sample. This packed bead columnis able to capture free MCP mAb or Fab flowed through the column. Theamount of free mAb or Fab in solution was determined using a secondarygoat anti-mouse heavy and light chain IgG-Cy5 conjugate.

A fixed amount of 11K2 mAb, 1A1 mAb, 11K2 Fab, or 1A1 Fab was incubatedwith various amounts of human MCP-1, MCP-2 or MCP-3 for three hours. Theamount of uncomplexed free antibody remaining in solution was determinedby flowing these mixtures over the MCP-1-loaded bead column and labelingwith the Cy5 secondary antibody. The fluorescent signal was plottedagainst the MCP concentration and the affinity was determined using aquadratic curve fit

Affinities determined for both 11K2 and 1A1 mAb and Fab molecules arelisted in Table 6 below. Exact affinities of 11K2 mAb and 1A1 mAb forhuman MCP-1 could not be determined, as the affinity is much lower thanthe lowest amount of antibody that can be detected by this method. Inthose cases, only an upper limit to the affinity can be determined.Affinity of the 11K2 Fab and 1A1 Fab for human MCP-3 was not determined(ND).

TABLE 8 MCP binding affinity measurements in solution Antibody MCP-1MCP-2 MCP-3 11K2 mAb  <4 × 10⁻¹³ M 1.8 × 10⁻¹¹ M >5 × 10⁻⁸ M 11K2 Fab1.1 × 10⁻¹¹ M 4.3 × 10⁻¹⁰ M ND 1A1 mAb  <7 × 10⁻¹³ M 1.2 × 10⁻¹² M >5 ×10⁻⁸ M 1A1 Fab 1.3 × 10⁻¹¹ M 3.2 × 10⁻¹⁰ M ND

In sum, monoclonal antibodies 1A1 and 11K2 recognize soluble human MCP-1and MCP-2 with a very high binding affinity which is in the low pMrange. Both 1A1 and 11K2 also recognize mouse MCP-1, while neitherrecognizes soluble MCP-3.

III. Cloning and Sequencing of 1A1 and 11K2 Monoclonal AntibodiesExample 9 Cloning and Sequencing of mu1A1 Variable Regions

Mouse monoclonal antibody 1A1 was cloned and sequenced according to thefollowing procedure. Total cellular RNA from 1A1 murine hybridoma cellswas prepared using the Qiagen RNeasy mini kit according to themanufacturer's recommended protocol.

cDNAs encoding the 1A1 variable regions of the heavy and light chainswere cloned by RT-PCR from total cellular RNA, following standardprocedures known to one of skill in the art. Briefly, following themanufacturer's recommended protocols, first-strand cDNAs (prepared withthe Amersham First-Strand cDNA Synthesis Kit) were amplified by PCRusing the Clontech Advantage 2 PCR Kit. The following primers were usedfor first-strand synthesis of the 1A1 heavy and light chain cDNAs(Y=C/T, and R=A/G): 1A1 Heavy Chain cDNA Primer: 5′-AGG TCT AGA AYC TCCACA CAC AGG RRC CAG TGG ATA GAC-3′ (SEQ ID NO: 3) and 1A1 Light ChaincDNA Primer: 5′-GCG TCT AGA ACT GGA TGG TGG GAG ATG GA-3′ (SEQ ID NO:4).

Primers used for PCR amplification of the murine 1A1 immunoglobulinheavy chain variable domain were as follows: 5′-AGG TSM ARC TGC AGS AGTCWG G-3′ (SEQ ID NO: 5) and 5′-TGA GGA GAC GGT GAC CGT GGT CCC TTG GCCCC-3′ (SEQ ID NO: 6) (S=C/G, M=A/C, R=A/G, and W=A/T). The primers usedfor PCR amplification of the murine 1A1 immunoglobulin light chainvariable domain were: 5′-GAY ATH CAR ATG AGN CAG-3′ (SEQ ID NO: 7) and5′-GCG TCT AGA ACT GGA TGG TGG GAG ATG GA-3′ (SEQ ID NO: 8) (Y=C/T,H=A/C/T, R=A/G, and N=A/C/G/T).

The PCR was performed at 30 cycles using Clontech's Advantage 2 PCR Kitusing the following PCR conditions: denature 0.5 min at 94° C., anneal 1min at 50° C., and elongate 1 min at 72° C. The PCR products weregel-purified using the Qiagen Qiaquick gel extraction kit following themanufacturer's recommended protocol. Purified 1A1 heavy and light chainPCR products were subcloned into Invitrogen's pCR2.1-TOPO vector usingtheir TOPO TA Cloning kit according to the manufacturer's recommendedprotocol (pCR-049=1A1 heavy chain, per-053=1A1 light chain). Insertsfrom multiple independent subclones were sequenced according to methodsknown in the art and those described in Sanger et al., PNAS 74,5463-5467, incorporated herein by reference, and subclones were found tobe identical.

Sequence data was analyzed according to BLAST analysis (seehttp://www.ncbi.nlm.nih.gov). Blast analyses of the variable domainsequences confirmed their immunoglobulin identity. The 1A1 heavy chainvariable domain was determined to be a member of murine subgroup II(C),while the 1A1 light chain variable region was determined to be a memberof murine kappa subgroup II. The predicted amino acid sequences of themature 1A1 murine variable domains, as well as the determined nucleotidesequences, are shown below in Tables 7 and 8.

TABLE 9 Nucleotide sequence of mu1A1 variable domains1A1 Heavy Chain Variable Region   1 GAGGTCCAGCTGCAGCAGTCTGGGGCAGAACTTGTGAGGTCAGGGGCCTCAGTCAAGTTG  60  61 TCCTGCACAGCTTCTGCCTTCAACATTAAAGACAACTATATGCACTGGGTGAAGCAGAGG 120 121 CCTGAACAGGGCCTGGAGTGGATTGGATGGATTGATCCTGAGAATGGAGATACTGAATAT 180 181 GCCCCGAAGTTCCAGGGCAAGGCCACTATGACTGCAGACACATCCTCCAACACAGCCTAC 240 241 CTGCAGCTCAGCAGCCTGACATCTGAGGACACTGCCGTCTATTACTGTAATACATGGGCT 300 301 TACTACGGTACTAGCTACGGGGGATTTGCTTACTGGGGCCAAGGGACCACGGTCACCGTC 360 361 TCCTCA (SEQ ID NO: 9) 366 1A1 Light Chain Variable Region   1GATATCCAGATGACTCAGACTCCACTCACTTTGTCGGTTACCATTGGACAACCAGCCTCC   60  61ATCTCTTGCAAGTCAAGTCAGAGCCTCTTAGATAGTGATGGAAAGACATATTTGAATTGG  120 121TCGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAACTGGAC  180 181TCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATC  240 241AGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGCTGGCAAGGTACACATTTTCCT  300 301CAGACGTTCGGTGGAGGCACCAAGCTGGAGATCAAA (SEQ ID NO: 10) 336

TABLE 10 Amino acid sequence of 1A1 variable domains(CDR domains underlined) 1A1 Heavy Chain Variable RegionEVQLQQSGAELVRSGASVKLSCTASGFNIKDNYMHWVKQRPEQGLEWIGW                              CDR1                    IDPENGDTEYAPKFQGKATMTADTSSNTAYLQLSSLTSEDTAVYYCNTWA     CDR2YYGTSYGGFAYWGQGTTVTVSS (SEQ ID NO: 11)  CDR31A1 Light Chain Variable RegionDIQMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWSLQRPGQSPK                           CDR1RLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFP    CDR2                                      CDR3QTFGGGTKLEIK (SEQ ID NO: 12)

Nucleotide and amino acid comparisons of the 1A1 variable heavy andlight chains are also shown in FIG. 7. The sequence of the CDR regionsof the 1A1 antibody were determined to be the following:

TABLE 11 CDRs of the 1A1 Antibody 1A1 Heavy Chain Variable Region CDR1:DNYMH (SEQ ID NO: 13) CDR2: WIDPENGDTEYAPKFQG (SEQ ID NO: 14) CDR3:WAYYGTSYGGFAY (SEQ ID NO: 15) 1A1 Light Chain Variable Region CDR1:KSSQSLLDSDGKTYLN (SEQ ID NO: 16) CDR2: LVSICLDS (SEQ ID NO: 17) CDR3:WQGTHFPQT (SEQ ID NO: 18)

Example 10 Cloning and Sequencing of mu11K2 Variable Regions

Mouse monoclonal antibody 11K2 was cloned and sequenced according to thefollowing procedure. Total cellular RNA from 11K2 murine hybridoma cellswas prepared using the Qiagen RNeasy mini kit according to themanufacturer's recommended protocol.

cDNAs encoding the variable regions of the heavy and light chains werecloned by RT-PCR from total cellular RNA. Following the manufacturersrecommended protocols, first-strand cDNAs (prepared with the AmershamFirst-Strand cDNA Synthesis Kit) were amplified by PCR using theClontech Advantage 2 PCR Kit. The following primers were used forfirst-strand synthesis of the 11K2 heavy and light chain cDNAs (Y=C/T,and R=A/G): 11K2 Heavy Chain cDNA Primer: 5′-AGG TCT AGA AYC TCC ACA CACAGG RRC CAG TGG ATA GAC-3′ (SEQ ID NO: 19) and 11K2 Light Chain cDNAPrimer: 5′-GCG TCT AGA ACT GGA TGG TGG GAG ATG GA-3′ (SEQ ID NO: 20).

The primers used far PCR amplification of the murine 11K2 immunoglobulinheavy chain variable domain were: 5′-GGG GAT ATC CAC CAT GGR ATG SAG CTGKGT MAT SCT CTT-3′ (SEQ ID NO: 21) and 5′ AGG TCT AGA AYC TCC ACA CACAGG RRC CAG TGG ATA GAC-3′ (SEQ ID NO: 22) (R=A/G, S=C/G, M=A/C, andY=C/T). The primers used for PCR amplification of the murine 11K2immunoglobulin light chain variable domain were: 5′-GAY ATH CAR ATG ACNCAG-3′ (SEQ ID NO: 23) and 5′-GCG TCT AGA ACT GGA TGG TGG GAG ATG GA-3′(SEQ ID NO: 24) (Y=C/T, H=A/C/T, R=A/G, and N=A/C/G/T).

The PCR was performed at 30 cycles using Clontech's Advantage 2 PCR Kitunder the following PCR conditions: denature 0.5 min at 94° C., anneal 1min at 50° C., and elongate 1 min at 72° C. The PCR products weregel-purified using the Qiagen Qiaquick gel extraction kit following therecommended protocol. Purified 11K2 heavy and light chain PCR productswere subcloned into Invitrogen's pCR2.1-TOPO vector using their TOPO TACloning kit according to the manufacturer's recommended protocol(pCR-008=11K2 heavy chain, per-033=11K2 light chain. Inserts frommultiple independent subclones were sequenced according to methods knownin the art and those described in Sanger et al., PNAS 74, 5463-5467,incorporated herein by reference, and the subclones were found to beidentical.

The variable light and heavy chains were compared with the consensussequences for mouse and human subgroups (Kabat et al, 1991) using theprogram FASTA and a database of consensus sequences, which are publiclyavailable (see http://people.cryst.bbk.ac.uk/˜ubcg07s/). The variablelight chain is a member of mouse subgroup Kappa 5 with a 68.8% identityin 113 aa overlap and the variable heavy chain is a member of mousesubgroup 2C with a 85.6% identity in 125 aa overlap. The variable lightchain corresponds to human subgroup Kappa 1 with a 69.9% identity in 113aa overlap. The variable heavy chain corresponds to human subgroup 1with a 59.7% identity in 129 aa overlap. The predicted amino acidsequences of the mature 11K2 murine variable domains, as well as thedetermined nucleotide sequences, are shown below in Tables 12 and 13.

TABLE 12 Nucleotide sequence of mu11K2 variable domains11K2 Heavy Chain Variable Region 1GAGGTTCAGCTGCAGCAGTCTGGGGCAGAGCTTGTGAAGGCAGGGGCCTCAGTCAAGTTG 60 61TCCTGCCCAGCTTGTGGCCTCAACATTAAAGACACCTATATGCACTGGGTGAAGCAGAGG 120 121CCTGAACAGGGCCTGGAGTGGATTGGAAGGATTGATCCTGCGAATGGTAATACTAAATTT 180 181GACCCGAAGTTCCAGGGCAAGGCCACTATAACAGCAGACACATCCTCCAACACAGCCTAC 240 241CTGCAGCTCAGCAGCCTGACATCTGAGGACACTGCCGTCTATTACTGTGCTAGAGGCGTC 300 301TTTGGCTTTTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA (SEQ ID NO: 25) 35111K2 Light Chain Variable Region 1GACATTCAGATGACTCAGTCTTCATCCTCCTTTTCTGTATCTCTAGGAGACAGAGTCACC 60 61ATTACTTGCAAGGCAACTGAGGACATATATAATCGATTAGCCTGGTATCAGCAGAAACCA 120 121GGAAGTGCTCCTAGGCTCTTAATTTCTGGTGCAACCAGTTTGGAGACTGGGGTTCCTTCA 180 181AGATTCAGTGGCAGTGGATCTGGAAAAGATTACACTCTCAGCATTACCAGTCTTCAGACT 240 241GAGGATGTTGCTACTTATTACTGTCAACAGTTTTGGAGTGCTCCGTACACGTTCGGAGGG 300 301GGGACCAAGCTGGAGATCAAA (SEQ ID NO: 26) 321

TABLE 13 Amino acid sequence of 11K2 variable domains(CDR regions underlined) 11K2 heavy chain variable regionEVQLQQSGAELVKAGASVKLSCPASGLNIKDTYMHWVKQRPEQGLEWIGR                              CDR1IDPANGNTKFDPKFQGKATITADTSSNTAYLQLSSLTSEDTAVYYCARGV      CDR2FGFFDYWGQGTTLTVSS (SEQ ID NO: 27) CDR3 11K2 light chain variable regionDIQMTQSSSSFSVSLGDRVTITCKATEDIYNRLAWYQQKPGSAPRLLISG                           CDR1                   ATSLETGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQFWSAPYTFGGCDR2                                    CDR3 GTKLEIK (SEQ ID NO: 28)

Nucleotide and amino acid comparisons of the 11K2 variable heavy andlight chains are also shown in FIG. 8. The sequence of the CDR regionsof the 11K2 antibody were determined to be as follows:

TABLE 14 CDRs of 11K2 Antibody 11K2 Heavy Chain Variable Region CDR1:DTYMH (SEQ ID NO: 29) CDR2: RIDPANGNTKFDPKFQG (SEQ ID NO: 30) CDR3:GVFGFFDY (SEQ ID NO: 31) 11K2 Light Chain Variable Region CDR1:KATEDIYNRLA (SEQ ID NO: 32) CDR2: GATSLET (SEQ ID NO: 33) CDR3:QQFWSAPYT (SEQ ID NO: 34)

Based on the results described above, particularly Tables 3 and 5,antibodies were grouped according to their antigen-binding specificity.The CDR region of mAbs which could recognize MCP-1, MCP-2, and MCP-3,including 4N4, 5A13, 6D21, 6I5, 7H1, 11K2, and 1A1 were determined asdescribed above. Sequencing revealed that mAbs 4N4, 5A13, 6D21, 6I5,7H1, and 11K2 all had identical sequences. Monoclonal antibody 1A1 had adifferent sequence. Thus, based on CDR cloning, as well as N-terminalsequencing, two distinct pan-MCP monoclonals antibodies were found toexist.

Example 11 PEGylated 11K2 Fab

As depicted in FIG. 13, PEGylation of the 11K2 Fab fragment does notinterfere with 11K2's in vitro activity. PEGylated 11K2 fragments weretested in a chemotaxis inhibition assay (as described previously), using20 ng·mL of MCP-1. PEGylated 11K2 fragments were as effective as the11K2 Fab alone at inhibiting migration of the cells.

Example 12 Chimeric 11K2 Antibody

The nucleotide and amino acid sequences of the 11K2 heavy chain chimericantibody are shown in FIG. 9A. The variable region is defined asnucleotides 1-351 (amino acids 1-117) of the heavy chain. The nucleotideand amino acid sequences of the 11K2 light chain chimeric antibody areshown in FIG. 9B, where the variable region is defined as nucleotides1-321 and amino acids 1-107.

IV. Humanized 11K2 Antibody Example 13 11K2 Humanization

Modeling the structure of the variable regions In order to identify keystructural framework residues in the murine 11K2 antibody, athree-dimensional model was generated based on the closest murineantibodies for the heavy and light chains The 11K2 light and heavychains were aligned against a local copy of the most recent PDB databaseto determine structural frames to be used to construct three dimensionalmodels of the light and heavy chains. Using FASTA the light chain wasfound to have 90.7% sequence identity to monoclonal murine antibody Fab184.1 (10SP; 1.8 Å), and the heavy chain was found to have 89.7%sequence identity to murine E8 Fab fragment (1WEJ; 1.8 Å).

Using the molecular modeling package Modeler 5.0 (Accelrys Inc) thethree dimensional structures of the light and heavy chains were builtusing antibodies 184.1 and E8, respectively. Five homology models werecreated, and the best one in terms of Modeler energy was selected.Procheck analysis showed that 1 residue (A51, light chain) was in adisallowed region of the phi/psi map, but by comparing phi/psi maps ofthe different models, the lowest energy model was also the best inphi/psi map violations.

Design of the reshaped variable regions Human germline sequences wereused as the acceptor frameworks for humanized 11K2. To find the closestgermline sequences, the NCBI NR database and the Kabat database weresearched for the most homologous expressed human frameworks in. In thissearch the CDR sequences were masked. The selection of the most suitableexpressed sequence includes checking for sequence identity of thecanonical and interface residues, and checking for the similarity in CDRloop lengths. The source of the antibody was also a determining factor.Previously humanized antibodies were excluded. For the NCBI NR databasesearch, a BLAST was used, and for the Kabat database search, FASTA wasused.

The most similar expressed light chain was found in the nr database(GI-486875; Griffiths et al. (1993), supra), and the most similar heavychain was found in the Kabat database (Kabat ID 000054; Kipps. & Duffy(1991), supra). Both sequences were searched against the database ofgermline sequences (http://www.ncbi.nlm.nih.gov/igblast/), and resultedin the following selected germlines: L11 for the light chain, and 1-69for the heavy chain.

As noted supra, the humanized antibodies of the invention comprisevariable framework regions substantially from a human immunoglobulin(acceptor immunoglobulin) and complementarity determining regionssubstantially from a mouse immunoglobulin (donor immunoglobulin) termed11K2. Having identified the complementarity determining regions of 11K2and appropriate human acceptor immunoglobulins, the next step was todetermine which, if any, residues from these components to substitute tooptimize the properties of the resulting humanized antibody. Thecriteria described supra were used to select residues for substitution.A summary of the backmutations is shown below in Table 15:

TABLE 15 Summary of backmutations of humanized 11K2 Backmutations inReshaped VL - Human germline L11 49 Y → S This residue is close to ahypervariable loop and it retained in both versions 69 T → K Thisresidue is close to a hypervariable loop but the sidechain is solventexposed. Only retained in the first version. 71 F → Y This is acanonical residue and is retained in both versions Backmutations inReshaped VH - Human germline 1-69 27 G → L This is a canonical residue.Retained in both versions. 28 T → N This residue is close to ahypervariable loop. Retained in first version. 29 F → I This residue isclose to a hypervariable loop. Retained in both versions. 30 S → K Thisresidue is close to a hypervariable loop. Retained in first version. 48M → I This residue is close to a hypervariable loop but is a fairlyconservative change. Retained in first version. 67 V → A This residue isclose to a hypervariable loop, and is close to residue 48. Retained infirst version. 73 K → T This residue is close to a hypervariable loop.Moreover, the human and mouse consensus is T. Retained in both versions.

FIG. 12 depicts alignments of the chimeric 11K2 VL and VH, respectively(same as the original murine 11K2 sequence), with the versions 1 and 2of the humanized 11K2 antibody. Two versions of the variable lightreshaped chain and two versions of the variable heavy reshaped chainwere designed. The first version contains the most backmutations and thesecond version contains the fewest (i.e. the most “humanized”). Thesequences of the two versions of each variable and heavy chains ofhumanized 11K2 are shown below:

Humanized 11K2 Heavy Chain (backmutations shown in lower case):

Version 1 (H1)(7 backmutations) (SEQ ID NO: 47)QVQLVQSGAEVKKPGSSVKVSCKASGlnikDTYMHWVRQAPGQGLEWiGRIDPANGNTKFDPKFQGRaTITADtSTSTAYMELSSLRSEDTAVYYCARGV FGFFDYWGQGTTVTVSSVersion 2 (H2)(3 backmutations) (SEQ ID NO: 48)QVQLVQSGAEVKKPGSSVKVSCKASGLTiSDTYMHWVRQAPGQGLEWMGRIDPANGNTKFDPKFQGRVTITADtSTSTAYMELSSLRSEDTAVYYCARGV FGFFDYWGQGTTNTIVSSHumanized 11K2 Light Chain (backmutations shown in lower case):

Version 1 (L1)(3 backmutations) (SEQ ID NO: 49)DIQMTQSPSSLSASVGDRVTITCKATEDIYNRLAWYQQKPGKAPKLLIsGATSLETGVPSRFSGSGSGkDyTLTISSLQPEDFATYYCQQFWSAPYTF GGGTKVEIKVersion 2 (L2) (2 backmutations) (SEQ ID NO: 50)DIQMTQSPSSLSASVGDRVTITCKATEDIYNRLAWYQQKPGKAPKLLIsGATSLETGVPSRFSGSGSGTDyTLTISSLQPEDFATYYCQQFWSAPYTF GGGTKVEIK

Tables 16 and 17 set forth Kabat numbering keys for the various lightand heavy chains of 11K2, respectively.

TABLE 16 Key to Kabat Numbering for 11K2 Heavy Chain Variable RegionMouse KABID Hum. Hum. Kabat # AA # Type 11K2 000054 11K2, v1 11K2, v2Comment  1 1 FR1 E Q Q Q  2 2 V V V V  3 3 Q Q Q Q  4 4 L L L L  5 5 Q VV V  6 6 Q Q Q Q  7 7 S S S S  8 8 G G G G  9 9 A A A A 10 10 E E E E 1111 L V V V 12 12 V K K K 13 13 K K K K 14 14 A P P P 15 15 G G G G 16 16A S S S 17 17 S S S S 18 18 V V V V 19 19 K K K K 20 20 L V V V 21 21 SS S S 22 22 C C C C 23 23 P K K K 24 24 A A A A 25 25 S S S S 26 26 G GG G 27 27 L G L L Canonical residue, retained 28 28 N T N T Residueclose to hypervariable loop, retained in v1 (H1) 29 29 I F I I Residueclose to hypervariable loop, retained 30 30 K S K S Residue close tohypervariable loop, retained in v1 (H1) 31 31 CDR1 D S D D 32 32 T Y T T33 33 Y A Y Y 34 34 M I M M 35 35 H S H H 36 36 FR2 W W W W 37 37 V V VV 38 38 K R R R 39 39 Q Q Q Q 40 40 R A A A 41 41 P P P P 42 42 E G G G43 43 Q Q Q Q 44 44 G G G G 45 45 L L L L 46 46 E E E E 47 47 W W W W 4848 I M I M Residue close to hypervariable loop, retained in v1 (H1) 4949 G G G G 50 50 CDR2 R G R R 51 51 I I I I 52 52 D I D D    52A 53 P PP P 53 54 A I A A 54 55 N F N N 55 56 G G G G 56 57 N T N N 57 58 T A TT 58 59 K N K K 59 60 F Y F F 60 61 D A D D 61 62 P Q P P 62 63 K K K K63 64 F F F F 64 65 Q Q Q Q 65 66 G G G G 66 67 FR3 K R R R 67 68 A V AV Residue close to hypervariable loop, retained in v1 (H1) 68 69 T T T T69 70 I I 1 I 70 71 T T T T 71 72 A A A A 72 73 D D D D 73 74 T E T TResidue close to hypervariable loop, retained 74 75 S S S S 75 76 S T TT 76 77 N S S S 77 78 T T T T 78 79 A A A A 79 80 Y Y Y Y 80 81 L M M M81 82 Q E E E 82 83 L L L L    82A 84 S S S S    82B 85 S S S S    82C86 L L L L 83 87 T R R R 84 88 S S S S 85 89 E E E E 86 90 D D D D 87 91T T T T 88 92 A A A A 89 93 V V V V 90 94 Y Y Y Y 91 95 Y Y Y Y 92 96 CC C C 93 97 A A A A 94 98 R R R R 95 99 CDR3 G G G G 96 100 V S V V 97101 F S F F 98 102 G W G G 99 103 F T F F 100  104 F F F F 101  105 D DD D 102  106 Y Y Y Y 103  107 FR4 W W W W 104  108 G G G G 105  109 Q QQ Q 106  110 G G G G 107  111 T T T T 108  112 T L T T 109  113 L V V V110  114 T T T T 111  115 V V V V 112  116 S S S S 113  117 S S S S

TABLE 17 Key to Kabat Numbering for 11K2 Light Chain Variable RegionMouse Hum. Hum. Kabat # AA # Type 11K2 GI-486875 11K2, v1 11K2, v2Comment 1 1 FR1 D D D D 2 2 I I I I 3 3 Q Q Q Q 4 4 M M M M 5 5 T T T T6 6 Q Q Q Q 7 7 S S S S 8 8 S P P P 9 9 S S S S 10 10 S S S S 11 11 F LL L 12 12 S S S S 13 13 V A A A 14 14 S S S S 15 15 L V V V 16 16 G G GG 17 17 D D D D 18 18 R R R R 19 19 V V V V 20 20 T T T T 21 21 I I I I22 22 T T T T 23 23 C C C C 24 24 CDR1 K R K K 25 25 A E A A 26 25 T S TT 27 27 E Q E E 28 28 D G D D 29 29 I I I I 30 30 Y R Y Y 31 31 N N N N32 32 R D R R 33 33 L L L L 34 34 A G A A 35 35 FR2 W W W W 36 36 Y Y YY 37 37 Q Q Q Q 38 38 Q Q Q Q 39 39 K K K K 40 40 P P P P 41 41 G G G G42 42 S K K K 43 43 A A A A 44 44 P P P P 45 45 R K K K 46 46 L L L L 4747 L L L L 48 48 I I I I 49 49 S Y S S Residue close to hypervariableloop, retained. 50 50 CDR2 G G G G 51 51 A T A A 52 52 T S T T 53 53 S SS S 54 54 L L L L 55 55 E Q E E 56 56 T S T T 57 57 FR3 G G G G 58 58 VV V V 59 59 P P P P 60 60 S S S S 61 61 R R R R 62 62 F F F F 63 63 S SS S 64 64 G G G G 65 65 S S S S 66 66 G G G G 67 67 S S S S 68 68 G G GG 69 69 K T K T Residue close to hypervariable loop, retained in v1 (L1)70 70 D D D D 71 71 Y F Y Y Canonical residue, retained. 72 72 T T T T73 73 L L L L 74 74 S T T T 75 75 I I I I 76 76 T S S S 77 77 S S S S 7878 L L L L 79 79 Q Q Q Q 80 80 T P P P 81 81 E E E E 82 82 D D D D 83 83V F F F 84 84 A A A A 85 85 T T T T 86 86 Y Y Y Y 87 87 Y Y Y Y 88 88 CC C C 89 89 CDR3 Q Q Q Q 90 90 Q Q Q Q 91 91 F T F F 92 92 W T W W 93 93S S S S 94 94 A F A A 95 95 P P P P 96 96 Y L Y Y 97 97 T T T T 98 98FR4 F F F F 99 99 G G G G 100 100 G G G G 101 101 G G G G 102 102 T T TT 103 103 K K K K 104 104 L V V V 105 105 E E E E 106 106 I I I I 107107 K K K K

Example 14 Cloning and Sequencing of Humanized 11K2

Four different humanized versions of 11K2 were made based oncombinations of two different humanized versions of the 11K2 heavy andlight chains. Germline sequences were chosen as human acceptorframeworks, including VK L11 for the light chain, and VH 1-69 for theheavy chain. The four combinations of humanized antibodies weredesignated H1-L1, H1-L2, H2-L1, and H2-L2. Amino acid sequences of thehumanized versions of the 11K2 heavy and light chains (nucleotide andamino acid) are shown in FIG. 10. An alignment of the heavy and lightchains of the 11K2 chimeric antibody and the humanized 11K2 antibody(versions 1 and 2) is shown in FIG. 11. The first version contains themost backmutations to the murine donor sequences, while the secondversion contains the fewest (i.e., the most “humanized”).

Hu11K2 variable regions were made by site-directed mutagenesis usingchimeric 11K2 variable domain plasmids as starting templates. Followingthe manufacturer's recommended protocol, the QuikChange Site-DirectedMutagenesis Kit (Stratagene Cat. #200518) was used to systematicallyintroduce (framework by framework) the residue changes outlined in abovein Tables 16 and 17, as well as FIG. 11. The mutagenic primers for theframework (FR) changes are described below. The cDNA sequence of thehuman acceptor frameworks (germline VK L11 for the light chain, andgermline VH 1-69 for the heavy chain) were used, with silent mutationsintroduced to produce restriction site changes to facilitateidentification of mutated plasmids. Mutated plasmids were identified byscreening for the introduced restriction site changes. The variableregion cDNA sequences in the resultant plasmids were confirmed by DNAsequencing.

Hu11K2 light chain mutagenesis was performed according to the followingprotocol. For the 11K2 version 1 light chain, plasmid pCR060 was used astemplate in a PCR reaction with the following primers: FR1 primer 5′ CCCGCG GAG ACA TTC AGA TGA CTC AGT CTC CAT CCT CCC TGT CAG CAT CTG TGG GAGACA GAG TCA CCA TTA CTT GCA AGG C 3′ (SEQ ID NO: 57), which added anAlwn1 site; FR2 primer 5′ GGT ATC AGC AGA AAC CAG GAA AGG CCC CTA AGCTCT TAA TTT CTG GTG CAA CC 3′ (SEQ ID NO: 58), which added an EcoO109 Isite; FR3 primer 5′ GGA AAA GAT TAC ACT CTC ACC ATT AGC AGT CTA CAG CCTGAG GAT TIT GCT ACT TAT TAC TGT CAA CAG 3′ (SEQ ID NO: 59), which addedan AccI site; and FR4 primer 5′ CGT TCG GAG GGG GGA CCA AGG TGG AGA TCTAAA AAA AGG GCG AAT TCT G 3′ (SEQ ID NO: 60), which added a StyI site.The resultant version 1 light chain plasmid was designated pCR067.

For the 11K2 version 2 light chain, plasmid pCR067 was used as templatein a PCR reaction with the following primer: FR3 primer 5′ GAT TCA GTGGCA GTG GAT CCG GAA CAG ATT ACA CTC TCA CCA TTA GC 3′ (SEQ ID NO: 61),which introduced a BspeI site. The resultant version 2 light chainplasmid was designated pCR06.

Hu11K2 heavy chain mutagenesis was performed according to the followingprotocol. For the 11K2 version 1 heavy chain, plasmid pCR046 was used astemplate with the following primers: FR1 primers 5′ GTG GTT ACA GGG GTCAAC TCA CAG GTT CAG CTG GTG CAG TCT GGG GCA GAG CTT G 3′ (SEQ ID NO:62), which added a Hinc2 site, and 5′ GCA GTC TGG GGC AGA GGT GAA GAAGCC CGG GTC CTC AGT CAA GGT CTC CTG CAA GGC TIC TGG CCT CAA CAT TAA AGAC 3′ (SEQ ID NO: 63), which added a Sma1 site; FR2 primer 5′ GAC ACC TATATG CAC TGG GTG CGA CAG GCG CCT GGA CAG GGC CTG GAG TGG ATT GG 3′ (SEQID NO: 64), which added a Nan site; FR3 primer 5′ CCC GAA GTT CCA GGGCAG AGC CAC TAT AAC AGC AGA CAC ATC CAC GAG CAC AGC CTA CAT GGA GCT CAGCAG CCT GAG ATC TGA GGA CAC TGC CG 3′ (SEQ ID NO: 65), which added aSac1 site; and FR4 primer 5′ GGG GCC AAG GGA CCA CTG TGA CAG TCT CCT CAGGTG AGT CCT AAG CTT GGT ACC CGG G 3′ (SEQ ID NO: 66), which added anAva2 site. The resultant version 1 heavy chain plasmid was designatedpCR066.

For the 11K2 version 2 heavy chain, plasmid pCR066 was used as templatein a PCR reaction with the following primers: FR1 primer 5′ GGT CTC CTGCAA GGC TTC AGG CCT CAC CAT TAG CGA CAC CTA TAT GCA CTG GG 3′ (SEQ IDNO: 67), which added a StuI site; FR2 primer 5′ GGC GCC TGG ACA GGG CCTCGA GTG GAT GGG AAG GAT TGA TCC TGC G 3′ (SEQ ID NO: 68), which added anXhoI site; and FR3 primer 5′ GAC CCG AAG TTC CAG GGC AGA GTC ACT ATA ACTGCA GAC ACA TCC ACG AGC ACA GCC 3′ (SEQ ID NO: 69), which added a PstIsite. The resultant version 2 heavy chain plasmid was designated pCR072.

Example 15 Expression of Humanized 11K2 Antibodies

Expression vectors for the hu11K2 light chains were made by subcloningthe 0.40 kb NotI-BglII light chain variable domain fragments frompCR067, pCR069 or pCR037 (murine 11K2 light chain variable domain bulkper reaction), and the 0.68 kb BclI-NotI fragment from the plasmidpEAG963, containing a human kappa light chain constant domain, into theNotI site of the pCEP4 EBV expression vector-derived plasmid pCH274,producing light chain expression vectors pCR068 (version 1), pCR077(version 2), and pCR045 (light chain chimera). NotI and BglII sites wereengineered onto pCR032 prior to digestion.

Expression vectors for the hu11K2 heavy chains were made by subcloningthe 0.49 kb NotI-HindIII heavy chain variable domain fragments frompCR066, pCR072 or pCR032* (murine 11K2 heavy chain variable domain), andthe 1.21 kb HindIII-NotI fragment from the plasmid pEAG964, containing ahuman IgG1 constant region, into the NotI site of the pCEP4 EBVexpression vector-derived plasmid pCH274, producing heavy chainexpression vectors pCR073 (version 1), pCR075 (version 2) and pCR054(heavy chain chimera). NotI and HindIII sites were engineered intoplasmid pCR032 prior to digestion.

Hu11K2 heavy and light chain expression vectors were co-transfected inall four (4) combinations (i.e. H1-L1, H1-L2, H2-L1 and H2-L2) into293-EBNA cells and transfected cells were tested for antibody secretionand specificity. Western blot analysis (developed with anti-human heavyand light chain antibodies) of whole cell lysates and the conditionedculture media indicated that hu11K2-transfected cells synthesized andefficiently secreted heavy and light chains at levels similar tochimeric 11K2-transfected cells. All combinations retained reactivity toMCP-1 (murine, primate, and human) and MCP-2 (human) immobilized onELISA plates.

Example 16 Characterization of Humanized 11K2 ELISA Screening

Binding specificities of chimeric 11K2 and the four versions ofhumanized 11K2 (H1-L1, H1-L2, H2-L1, and H2-L2) were determined byELISA, according to the protocol described in Example 1. Human, mouse,and primate MCP-1 were used as antigen, and antibody 3G9, which does notbind MCP-1, was used as a negative control. ELISA plates (Corning, Inc.Cat#3369) were coated with 50 ng MCP-1 per well overnight at 4° C.Plates were washed then blocked with PBS/5% milk for 1.5 hrs at 25° C.Another wash was followed by incubation with the 11K2 mAbs (or 309isotype control) at the concentrations indicated for 1 hr at 25° C.After washing, the plates were incubated with secondary antibody(anti-human IgG-HRP, Jackson Immunoresearch Cat#109-036-088) for 1 hr at25° C. Plates were then washed and developed using the ABTS PeroxidaseSubstrate Kit (Vector Laboratories Cat#SK-4500). OD (405 nm) values wereplotted as a function of 11K2 mAb concentration.

Results from the ELISA experiment are shown below in Table 18 and FIGS.14A and 14B. These results demonstrate that each of the humanized andchimeric antibodies retained MCP-1 reactivity for each of the speciestested.

TABLE 18 Summary of humanised 11K2 reactivities MCP-1 MCP-2 MCP-3 HumanMouse Monkey Human Human Chimera yes yes yes yes no H1-L1 yes yes yesyes no H1-L2 yes yes yes yes no H2-L1 yes yes yes yes no H2-L2 yes yesyes yes no

Binding Affinity Measurement of Humanized 11K2

Binding affinity studies were performed in order to compare the fourversions of the humanized 11K2 anti-MCP antibody to chimeric and murine11K2 antibodies. KinExA experiments were performed, as previouslydescribed. Briefly, reagents that were used included the following.Recombinant human MCP-1 (catalog #279-MC) and MCP-2 (catalog #281-CP,R&D Systems, Inc.) were reconstituted to 100 μg/ml in PBS 0.1% BSA andstored at 4° C. for the duration of the experiment mu11K2 was purifiedfrom hybridoma cell line supernatant. Humanized 11K2 was purified from293 cell supernatant. All Fab fragments were generated according tostandard methods. NHS Sepharose beads (catalog #17-0906-01), AmershamBiosciences (Uppsula, Sweden) were also used to determine affinitythrough the KinExA method. Goat anti-human Cy-5 conjugate (catalog#109-177-003, Jackson ImmunoResearch) was also used.

To determine the binding affinity of the hu11K2, NHS-Sepharose beadswere washed six times in dH₂O and once in 50 mM NaHCO₃, pH 9.5. 1 ml ofMCP-1 at a concentration of 10 μg/ml in NaHCO₃ was added to an amount ofbeads equivalent to 1 ml of the original slurry. The beads were rockedovernight at 4° C. Beads were spun down and washed once with 1M Tris, pH8.0, then resuspended in 1M Tris pH 8.0 with 1% BSA, 0.02% azide. Thebeads were rocked at room temperature for one hour then stored at 4° C.in this buffer for the duration of the experiments.

The KinExA 3000 measures protein-protein interaction in solution, and isideally suited for measuring affinities of antibodies to antigens. Inthe typical experiment, antigen (MCP) is immobilized on a bead, antibodyis flowed over a column of these beads at a given concentration, and theantibody that binds to the beads is detected using a fluorescentsecondary. Antibody at a fixed concentration is then incubated withdifferent concentrations of antigen in solution and the amount of freeantibody is measured by flowing the mixture over the bead column quicklyenough that no re-equilibration occurs between solution and solid phase.The amount of antibody remaining free is plotted against theconcentration of antigen added and the data are fit to a quadraticequation to determine the affinity of the interaction in solution.

In these experiments, the only difference to the typical experimentdescribed above is that instead of using intact antibody, a Fab fragmentof 11K2 was used. Earlier KinExA experiments using the intact murine11K2 mAb binding to MCP-1 and MCP-2 gave biphasic curves that thesoftware could not interpret. This may be due to these proteins actingas dimers, so to eliminate the dimer/dimer interaction we generated Fabfragments. The binding of the 11K2 Fab fragments to MCP-1 and MCP-2produced curves that the KinExA software could fit. Results from theKinExA experiment are shown below in Table 19.

TABLE 19 MCP affinity measurements in solution of humanized 11K2 Fabvariants Antibody MCP-1 MCP-2 MCP-3 mu11K2 Fab  11 pM 430 pM >50 nMCh11K2 19.6 pM 226 pM ND H1L1 36.4 pM 725 pM* ND H2L2  77.5 pM* 3.2 nM*ND H1L2 62.3 pM 712 pM* ND H2L1 64.0 pM 940 pM* ND 1A1 Fab 12.9 pM 320pM >50 nM D9 mAb 41.5 pM ND ND S14 mAb (commercial) <5.6 pM ND ND*significant difference from murine and chimeric

Example 17 Inhibition of THP-1 Chemotaxis by Chimeric and Humanized 11K2

Chemotaxis inhibition assays were performed as previously described inExample 3, using MCP-1 to induce THP-1 cell migration. MCP-1 was used ata concentration of 2.3 nM. The results, shown in FIG. 12A, demonstratethat chimeric 11K2 and humanized versions were as effective asmonoclonal 11K2 at inhibiting THP-1 cell chemotaxis. In additionalexperiments using MCP-2 (at a concentration of 56 nM) to induce THP-1migration, chimeric and all humanized versions of 11K2 were effective atinhibiting chemotaxis (FIG. 12). Interestingly, the agonist effectobserved with monoclonal 11K2 was also observed with chimeric andhumanized 11K2 versions, as to seen in FIG. 12B. In contrast to forms of11K2 mAb having normal Fc region functionality, an aglycosylated form ofthe chimeric 11K2 mAb no longer demonstrates agonist activity towardshuman MCP-2. Thus, the aglycosylated form of 11K2 is acting as acomplete antagonist of both human MCP-1 and MCP-2.

V. Humanized 1A1 Antibody Example 18 1A1 Humanization

Modeling the structure of the variable regions In order to identify keystructural framework residues in the murine 1A1 antibody, athree-dimensional model was generated based on the closest murineantibodies for the heavy and light chains. The 1A1 light and heavychains were aligned against a local copy of the most recent PDB databaseto determine structural frames to be used to construct three dimensionalmodels of the light and heavy chains. Using FASTA the light chain wasfound to have 91.1% sequence identity to monoclonal murine antibodyFab1583 (1NLD; 2.9 Å), and have 87.4% sequence identity to the catalyticmurine antibody D2.5 (1YEE; 2.2 Å). The heavy chain was found to have85.7% sequence identity to murine 2E8 Fab fragment (12E8; 1.9 Å), and68.1% sequence identity to the murine monoclonal antibody F9.13.7 (1FBI;3.0 Å). The reason for including 1FBI with a relatively low sequencehomology was that it has the same H3 loop length and also a relativelyhigh sequence conservation in the H3 loop.

Two composite antibody structures were created by aligning the lightchains of 1NLD and 1YEE onto the light chains of 12E8 and 1FBI,respectively. The atoms used to define the superposition were the Caatoms of the light chain interface residues. The two full structuraltemplates were obtained by saving the structural combinations of1NLD(light)/12E8(heavy) and 1YEE(light)/1FBI(heavy).

Using the molecular modeling package Modeler 5.0 (Accelrys Inc.) thethree dimensional structures of the light and heavy chains were builtusing the two composite structures. Five homology models were created,and the best one in terms of Modeler energy was selected. Procheckanalysis showed that no residues were in a disallowed region of thephi/psi map.

Design of the reshaped variable regions Human germline sequences wereused as the acceptor frameworks for humanized 1A1. To find the closestgermline sequences, the NCBI NR database and the Kabat database weresearched for the most homologous expressed human frameworks in. In thissearch the CDR sequences were masked. The selection of the most suitableexpressed sequence included checking for sequence identity of thecanonical and interface residues, and checking for the similarity in CDRloop lengths. The source of the antibody was also a determining factor.Previously humanized antibodies were excluded. BLAST was used for theNCBI NR database search, and the Kabat database was used for the FASTAsearch.

The most similar expressed light chain was found in the nr database(GI-284256; Kennedy (1991), supra), and the most similar heavy chain wasfound in the Kabat database (Kabat ID 037655; Bejcek et al. (1995)supra). Both sequences were searched against the database of germlinesequences (http://www.ncbi.nlm.nih.gov/igblast/), which resulted in thefollowing selected germlines: A17 for the light chain, and 5-51 for theheavy chain. The light chain germline A17 was identical to the expressedsequence GI-284256 in the framework regions. There were many sequencedifferences between the 5-51 germline and the Kabat ID 037655 expressedheavy chain, therefore the expressed sequence (25C1) was used instead ofthe closest germline for the heavy chain.

As noted supra, the humanized antibodies of the invention comprisevariable framework regions substantially from a human immunoglobulin(acceptor immunoglobulin) and complementarity determining regionssubstantially from a mouse immunoglobulin (donor immunoglobulin) termed1A1. Having identified the complementarity determining regions of 1A1and appropriate human acceptor immunoglobulins, the next step was todetermine which, if any, residues from these components to substitute tooptimize the properties of the resulting humanized antibody. Thecriteria described supra were used to select residues for substitution.A summary of the backmutations is shown below in Table 20:

TABLE 20 Summary of backmutations of humanized 1A1 Backmutations inreshaped VL - Human germline A17  2 V −> I This is a canonical residueand is retained in both versions. 36 F −> L This is an interface residueand a significant change. Retained in both versions. 45 R −> K This is asurface residue but is close to a hypervariable loop. Retained in firstversion. Backmutations in reshaped VH - Expressed sequence 25C1 (closestgermline 5-51) 27 Y → F This is a canonical residue, but a conservativechange. Retained in first version. 28 A → N This residue is close to ahypervariable loop. Retained in first version. 29 F → I This residue isa canonical residue. Retained in both versions. 30 S → K This residue isclose to a hypervariable loop. Retained in both versions. 66 Q → K Thisresidue is close to a hypervariable loop and interacts with an Asp.Retained in first version. 69 L → M This residue is close to ahypervariable loop but is a conservative change. Retained in firstversion. 73 K → T This residue is close to a hypervariable loop,contacting an acidic residue. Retained in both versions. 76 S → N Thisresidue is close to a hypervariable loop. Retained in first version. 91S → Y This residue is an interface residue and a big change. Retained inboth versions. 93 A → N This residue contacts a hypervariable loop.Retained in both versions. 94 R → T This residue is a canonical residue.Retained in both versions.

Two versions of the variable light reshaped chain and two versions ofthe variable heavy reshaped chain were designed. The first versioncontains the most backmutations and the second version contains thefewest (i.e. the most “humanized”). The sequences of the two versions ofeach variable and heavy chains of humanized 1A1 are shown below:

Humanized 1A1 Heavy Chain (backmutations shown in lower case):

Version 1 (11 backmutations) (SEQ ID NO: 53)QVQLLESGAELVRPGSSVKISCKASGfnikDNYMHWVKQRPGQGLEWIGWIDPENGDTEYAPKFQGkATmTADtSSnTAYMQLSGLTSEDSAVYyCntWA YYGTSYGGFAYWGQGTTVTVersion 2 (6 backmutations) (SEQ ID NO: 54)QVQLLESGAELVRPGSSVKISCKASGYAikDNYMHWVKQRPGQGLEWIGWIDPENGDTEYAPKFQGQATLTADtSSSTAYMQLSGLTSEDSAVYyCntWA YYGTSYGGFAYWGQGTTVTHumanized 1A1 Light Chain (backmutations shown in lower case):

Version 1 (3 backmutations) (SEQ ID NO: 55)DiVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWlQQRPGQSPkRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFP QTFGQGTKLEIKVersion 2 (2 backmutations) (SEQ ID NO: 56)DiVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWlQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFP FPQTFGQGTKLEIK

Tables 21 and 22 set forth Kabat numbering keys for the various lightand heavy chains of 1A1, respectively.

TABLE 21 Key to Kabat Numbering for 1A1 Heavy Chain Variable Region Hum.Hum. Kabat # AA # Type Mouse 1A1 25C1 1A1, v1 1A1, v2 Comment  1 1 FR1 EQ Q Q  2 2 V V V V  3 3 Q Q Q Q  4 4 L L L L  5 5 Q L L L  6 6 Q E E E 7 7 S S S S  8 8 G G G G  9 9 A A A A 10 10 E E E E 11 11 L L L L 12 12V V V V 13 13 R R R R 14 14 S P P P 15 15 G G G G 16 16 A S S S 17 17 SS S S 18 18 V V V V 19 19 K K K K 20 20 L I I I 21 21 S S S S 22 22 C CC C 23 23 T K K K 24 24 A A A A 25 25 S S S S 26 26 G G G G 27 27 F Y YF Canonical residue, retained in v1 (H1) 28 28 N A A N Residue close tohypervariable loop, retained in v1 (H1) 29 29 I F I I Canonical residue,retained 30 30 K S K K Residue close to hypervariable loop, retained 3131 CDR1 D S D D 32 32 N Y N N 33 33 Y W Y Y 34 34 M M M M 35 35 H N H H36 36 FR2 W W W W 37 37 V V V V 38 38 K K K K 39 39 Q Q Q Q 40 40 R R RR 41 41 P P P P 42 42 E G G G 43 43 Q Q Q Q 44 44 G G G G 45 45 L L L L46 46 E E E E 47 47 W W W W 48 48 I I I I 49 49 G G G G 50 50 CDR2 W Q WW 51 51 I I I I 52 52 D Y D D    52A 53 P P P P 53 54 E G E E 54 55 N DN N 55 56 G G G G 56 57 D D D D 57 58 T T T T 58 59 E N E E 59 60 Y Y YY 60 61 A N A A 61 62 P G P P 62 63 K K K K 63 64 F F F F 64 65 Q K Q Q65 66 G G G G 66 67 FR3 K Q Q K Residue close to hypervariable loop,retained in v1 (H1) 67 68 A A A A 68 69 T T T T 69 70 M L L M Residueclose to hypervariable loop, retained in v1 (H1) 70 71 T T T T 71 72 A AA A 72 73 D D D D 73 74 T K T T Residue close to hypervariable loop,retained 74 75 S S S S 75 76 S S S S 76 77 N S S N Residue close tohypervariable loop, retained in v1 (H1) 77 78 T T T T 78 79 A A A A 7980 Y Y Y Y 80 81 L M M M 81 82 Q Q Q Q 82 83 L L L L    82A 84 S S S S   82B 85 S G G G    82C 86 L L L L 83 87 T T T T 84 88 S S S S 85 89 EE E E 86 90 D D D D 87 91 T S S S 88 92 A A A A 89 93 V V V V 90 94 Y YY Y 91 95 Y S Y Y Residue is an interface residue, retained. 92 96 C C CC 93 97 N A N N Residue contacts hypervariable loop, retained. 94 98 T RT T Residue is canonical, retained 95 99 CDR3 W K W W 96 100 A T A A 97101 Y I Y Y 98 102 Y S Y Y 99 103 G S G G 100  104 T V T T  100A 105 S VS S  100B 106 Y D Y Y  100C 107 G F G G  100D 108 G Y G G 100E 109 F F FF 101  110 A D A A 102  111 Y Y Y Y 103  112 FR4 W W W W 104  113 G G GG 105  114 Q Q Q Q 106  115 G G G G 107  116 T T T T 108  117 T T T T109  118 V V V V 110  119 T T T T

TABLE 22 Key to Kabat Numbering for 1A1 Light Chain Variable RegionMouse Hum. Hum. Kabat # AA # Type 1A1 GI-284256 1A1, v1 1A1, v2 Comment 1 1 FR1 D D D D  2 2 I V I I Canonical residue, retained  3 3 Q V V V 4 4 M M M M  5 5 T T T T  6 6 Q Q Q Q  7 7 S S S S  8 8 S P P P  9 9 SL L L 10 10 S S S S 11 11 F L L L 12 12 S P P P 13 13 V V V V 14 14 S TT T 15 15 L L L L 16 16 G G G G 17 17 Q Q Q Q 18 18 P P P P 19 19 A A AA 20 20 S S S S 21 21 I I I I 22 22 S S S S 23 23 C C C C 24 24 CDR1 K RK K 25 25 S S S S 26 25 S S S S 27 27 Q Q Q Q    27A 28 S S S S    27B29 L L L L    27C 30 L V L L    27D 31 D Y D D   27E 32 S S S S 28 33 DD D D 29 34 G G G G 30 35 K N K K 31 36 T T T T 32 37 Y H Y Y 33 38 L LL L 34 39 N N N N 35 40 FR2 W W W W 36 41 L F L L Interface residue,retained. 37 42 L Q Q Q 38 43 Q Q Q Q 39 44 R R R R 40 45 P P P P 41 46G G G G 42 47 Q Q Q Q 43 48 S S S S 44 49 P P P P 45 50 K R K R Surfaceresidue close to hypervariable loop, retained in v1. 46 51 R R R R 47 52L L L L 48 53 I I I I 49 54 Y Y Y Y 50 55 CDR2 L K L L 51 56 V V V V 5257 S S S S 53 58 K N K K 54 59 L R L L 55 60 D D D D 56 61 S S S S 57 62FR3 G G G G 58 63 V V V V 59 64 P P P P 60 65 D D D D 61 66 R R R R 6267 F F F F 63 68 T S S S 64 69 G G G G 65 70 S S S S 66 71 G G G G 67 72S S S S 68 73 G G G G 69 74 T T T T 70 75 D D D D 71 76 F F F F 72 77 TT T T 73 78 L L L L 74 79 K K K K 75 80 I I I I 76 81 S S S S 77 82 R RR R 78 83 V V V V 79 84 E E E E 80 85 A A A A 81 86 E E E E 82 87 D D DD 83 88 L V V V 84 89 G G G G 85 90 V V V V 86 91 Y Y Y Y 87 92 Y Y Y Y88 93 C C C C 89 94 CDR3 W M W W 90 95 Q Q Q Q 91 96 G G G G 92 97 T T TT 93 98 H H H H 94 99 F W F F 95 100 P P P P 96 101 Q Y Q Q 97 102 T T TT 98 103 FR4 F F F F 99 104 G G G G 100  105 G Q Q Q 101  106 G G G G102  107 T T T T 103  108 K K K K 104  109 L L L L 105  110 E E E E 106 111 I I I I 107  112 K K K K

VI. In Vivo Efficacy of Anti-MCP Antibody Example 19 Efficacy ofAnti-MCP Antibody Treatment in TNBS-Induced Murine Colitis Model

To determine the efficacy of anti-MCP antibodies in treatinginflammatory disorders, a mouse model of colitis was selected. Colitiswas induced in Balb/c mice as previously described (Neurath et al.(1995) J Exp Med. 182(5):1281). Briefly, 6-8 week old female Balb/c mice(Charles River, Monza, Italy) were fasted for 1 day, anesthetized, and a3.5 F catheter was inserted into the colon such that the tip was 4 cmproximal to the anus. To induce colitis in the experimental mice, 1.0 mgof TNBS (Sigma Chemical Co, St Louis, Mo.) in 50% ethanol wasadministered via catheter into the lumen using a 1 ml syringe (injectionvolume of 100 μl). Control mice received 50% ethanol alone.

Following induction of colitis, mice were monitored daily for appearanceof diarrhea, loss of body weight, and survival. At the end of theexperiment, surviving mice were sacrificed and blood samples collectedby cardiac puncture. A 7 cm segment of colon was excised, weighed, andevaluated for macroscopic damage. Tissue segments were then used forimmunohistochemical studies, or were homogenized in protein extractionbuffer (Pierce, Rockford, Ill. USA) for use in cytokine andmyeloperoxidase (MPO) activity measurements as described (Fiorucci etal. (2002) Immunity 17: 769). Chemokine measurements were performedusing a commercially available ELISA assay for MCP-1 (R+D Systems,Minneapolis, Minn. USA).

TMS-induced colitis mice receiving 11K2 were studied for physicalchanges (e.g., weight loss), reduction in proinflammatory mediators, andreduction in circulating MCP-1 to determine the efficacy of 11K2 attreating colitis. TNBS-induced colitis experiments using different formsof the 11K2 antibody were performed in parallel with control antibodymouse monoclonal antibody MOPC21.

Anti-MCP Antibody Treatment Prevents Weight Loss and Enhances Survival

Colitis was induced in experimental mice as described above.Intraperitoneal (IP) injection of monoclonal antibody 11K2 and thecontrol antibody (IgG1b antisera MOPC21) was performed on days −1, 2 and5. Mice were administered either 200 μg of mouse monoclonal antibody11K2 or mouse control monoclonal antibody MOPC21. Mice were monitoredfor weight gain/loss and survival for seven days. On day 1, all micewere observed to weigh about 18 grams. Rapid weight gain was observed incontrol mice, which reached a plateau weight of about 23 grams by day 4of the trial. While weight gain in colitis model mice administeredmonoclonal antibody 11K2 was initially not as rapid as for control mice(non-colitis induced mice), gradual weight gain was observed in the miceover the course of the trial, reaching about 22 grams by day 7. Incontrast, TNBS-induced colitis model mice that received either controlmonoclonal antibody MOPC21 or no antibody failed to gain significantweight between days 1 and 7 of the trial, continuing to weigh about 18grams on day 7. Thus, 11K2 treated mice showed significant weight gain.

Treatment of colitis-induced mice with 11K2 also improved survival overthe seven day course of the trial, as compared to mice treated with TNBSalone or the combination of TNBS and the control monoclonal antibodyMOPC21. As shown in FIG. 15, about 70% of colitis model mice treatedwith 11K2 survived the seven day trial, in contrast to a survival rateof about 40% for colitis model mice administered either the controlmonoclonal antibody or no antibody. Mouse monoclonal antibody 11K2treatment therefore reduced lethality associated with TNBS-inducedcolitis in model mice.

Reduction of Proinflammatory Mediators in Anti-MCP Antibody-Treated Mice

To determine the effect of anti-MCP antibodies at reducing moleculesassociated with inflammation, colon tissues from TNBS-induced colitismice were dissected and assayed for concentration of proinflammatorymediator cytokines, TNFα, IFN-γ, and IL-2 according to manufacturer'sprotocols (R+D Systems, Minneapolis, Minn. USA). Concentrations of thethree cytokines were observed to be less than 100 μg/mg in control micethat were not administered TNBS. TNBS induction elevated colonic levelsof all three assayed cytokines in TNBS-induced mice and miceadministered the combination of TNBS and control monoclonal antibodyMOPC21, wherein about 600 pg/mg TNFα, 750 pg/mg IFN-γ, and 500 pg/mgIL-2 was observed. Lower levels of TNFα, IFN-γ, and IL-2 (about 200pg/mg, 300 pg/mg and 200 pg/mg, respectively) were observed in colontissues obtained from TNBS-induced colitis mice injected with 11K2.Thus, 11K2 blockade of MCP-1 decreased production of proinflammatorymediators in the inflamed colon.

Reduction of Circulating MCP-1 Levels in Anti-MCP Antibody-Treated Mice

To determine the effect of 11K2 on MCP-1 in TNBS-induced colitis modelmice, circulating levels of MCP-1 were analyzed according tomanufacturer's protocols (R+D Systems, Minneapolis, Minn. USA). As shownin FIG. 16, MCP-1 was observed at about 500 pg/ml serum in control mice,whereas serum MCP-1 levels in TNBS-induced colitis mice administeredcontrol monoclonal antibody MOPC21 or no antibody were about 3000 pg/mlserum. MCP-1 levels in TNBS-induced colitis mice treated with 11K2 wereobserved to be about 1000 pg/ml serum, representing a significantreduction of circulating MCP-1 levels relative to TNBS-induced colitismice administered the control monoclonal antibody or left untreated.Thus, 11K2 treatment significantly reduced the level of MCP-1 incirculation during colitis.

Dose-Dependent Analysis of Anti-MCP Antibody Treatment

Body weight was monitored for groups of TNBS-induced colitis miceinjected with varying amounts of mouse 11K2 monoclonal antibody todetermine dosage response. 11K2 was administered at doses of 2, 50, 100,and 200 μg/mouse three times a week, respectively. TNBS-induced colitiscontrol mice that did not receive antibody injections or colitis micereceiving only 2 μg doses of 11K2 showed a decline in weight about 25%relative to starting weights over the course of the experiment. Improvedbody weights were observed for mice administered 50 μg doses of 11K2(weight declines of about 15%), while improved body weights wereobserved for mice administered 100 μg and 200 μg doses (weights of thesemice were observed to be about equal to starting weights, with slightweight gain observed for those mice treated with 200 μg of 11K2).Uninduced and untreated control mice gained about 15% of body weightover the course of the experiment.

The effect of various doses of 11K2 was also studied by analyzingmyeloperoxidase (MPO) activity levels in TNBS-induced colitis mice. Toassess MPO activity levels, 50 μl SureBlue TMB (Kirkegaard & PerryLaboratories, Inc.) was added to 50 μl sample, e.g., serum or colonhomogenate. This mixture was allowed to incubate at room temperature for5 minutes, with 100 μl 0.18M H₂SO₄ then added to the reaction mixture.Absorbance at 450 nm was detected on a plate reader for all samples,with a range of 0.25 to 1 activity unit per sample. A standard curve wasgenerated using purified MPO (Sigma) in the peroxidase assay. MPOactivity levels were ascribed to samples by comparing detectedperoxidase activity values with the standard curve. MPO activity inuninduced, untreated control mice was observed to be about 15 U/mg.TNBS-induced mice left untreated exhibited about 35 U/mg MPO activity.Reduced levels of MPO activity were observed in TNBS-induced colitismodel mice that had been treated with 50 μg, 100 μg and 200 μg doses of11K2, with MPO activity levels observed to be about 25 U/mg, 18 U/mg,and 15 U/mg, respectively.

Efficacy of Humanized Anti-MCP Antibody and Pegylated Fab in TreatingColitis

To determine the efficacy of humanized 11K2 and 11K2 pegylated-Fab 11K2PEG-Fab), TNBS-induced mice were treated via intraperitoneal (IP)injection with mouse monoclonal antibody 11K2, humanized 11K2 (h11K2)antibody, chimeric 11K2 antibody, aglycosylated 11K2, or no antibody asa control. TNBS-induced colitis model mice were monitored for MPOactivity levels. As shown in FIG. 17, administration of either hu11K2(FIG. 17A) or 11K2 PEG-Fab (FIG. 17B) to TNBS-induced colitis miceresulted in significantly lower MPO levels than TNBS-induced colitiscontrol mice. Both hu11K2- and 11K2 PEG-Fab-treated mouse MPO levelswere comparable to those observed for TNBS-induced colitis mice treatedwith the mouse monoclonal antibody 11K2.

Therapeutic Treatment of Colitis Model Mice by Anti MCP Antibody

TNBS was administered to groups of mice and colitis was allowed toprogress for seven days. Mouse monoclonal antibody 11K2 or a controlmonoclonal antibody were then administered via IP injection to the miceon day 7. Body weight was then measured on days 10 and 14. In addition,MCP-1, TNFα, and MPO activity levels were assessed on day 14 for allmice. Elevated body weight, and significantly inhibited levels of MCP-1,TNFα and MPO activity were all observed for colitis-induced mice thathad been treated with 11K2 antibody (see FIG. 18), as compared tocolitis model mice left untreated or administered the non-therapeuticcontrol monoclonal antibody.

Example 20 Efficacy of Anti-MCP Antibodies in Atherosclerosis

To determine the efficacy of 11K2 at treating atherosclerosis, themurine ApoE-deficient model was used. Administration of mouse monoclonalantibody 11K2 to atherosclerotic model mice (apoE-deficient) wasperformed according to the methods of Lutgens et al. (2000) Proc. Natl.Acad. Sci. USA. 97:7464). ApoE −/− mice (Iffa Credo) were fed normalchow diet, and administered either 11K2 or a mouse control monoclonalantibody (IgG1b antisera MOPC21) at 200 μg per mouse by intraperitonealinjection twice a week for 12 weeks. Injection of the early treatmentgroup started at 5 weeks of age (n=15 11K2 antibody; n=15 controlantibody), when hardly any atherosclerotic lesions are observed to bepresent. Injections of the delayed treatment group (n=15 11K2 antibody;n=15 Control antibody) started at 17 weeks of age, at which pointadvanced atherosclerotic plaques are known to have developed. Mice ofboth early and delayed treatment groups were sacrificed at the end of 12weeks of treatment for examination of plaque development.

Atherosclerotic plaques were divided into initial and advanced lesions.Initial lesions were defined as fatty streaks containingmacrophage-derived foam cells with intracellular lipid accumulation (AHAtype II) or pools of extracellular lipid (AHA type III), whereasadvanced lesions contained extracellular lipid, a lipid core (AHA typeIV), and/or a fibrous cap (AHA type Va-c) (Stary et al. (1995)Arterioscler. Thromb. Vasc. Biol. 15:1512). Tissue processing,histological classification, and morphometry were performed as describedpreviously (Lutgens et al. (1999) Nat. Med. 5:1313; Lutgens et al.(1999) Circulation 99:276). 11K2 antibody-treated apoE −/− mice werecompared with control-treated apoE −/− mice. 11K2 antibody-treated apoE−/− mice of the delayed treatment group were also compared withcontrol-treated 17-wk-old apoE −/− mice to investigate plaqueprogression after treatment. For all analyses, a nonparametricMann-Whitney U test was used. The level of statistical significance wasset at P=0.05. As shown in FIG. 19, significant reduction of totalplaque area in the aortic arch was observed for both early and advancedgroups of mice treated with 11K2.

Quantities of atherosclerotic plaques classified as initial and advancedlesions were analyzed for all groups of mice. As shown in FIG. 19,treatment of atherosclerotic mice with 11K2 reduced the number ofplaques. Reduction in the total number of plaques observed in the earlytreatment group was comparable between control and experimental groupswhile about 5 plaques were observed in the aortic arches of both MOPC21(IgG1b control antisera)- and 11K2-administered mice. Advanced lesioncounts were lower for delayed treatment group mice treated with 11K2(about 3 advanced lesions per aortic arch observed), as compared todelayed treatment group MOPC21-treated mice (about 4 advanced lesionsper aortic arch observed). Too few advanced lesion plaques were observedin mice of the early treatment group to assess whether 11K2 treatmentreduced advanced lesion formation in the early treatment groups of mice.

All mice were also examined for both macrophage content and CD45+ cellcontent. Sections were immunolabeled with ED-²⁰ (1:10) for the detectionof macrophages or anti-CD45 antibody. Atherosclerosis model mice treatedwith 11K2 exhibited reductions in macrophage content of both initial andadvanced lesions, among both early and delayed treatment groups of mice(about 75% versus about 85% macrophage content for initial lesions inthe early treatment group; about 70% versus about 80% macrophage contentfor initial lesions of the delayed treatment group; and about 45% versusabout 55% macrophage content for advanced lesions of the delayedtreatment group). When CD45+ cell content was examined, advanced lesionsof the delayed treatment group were significantly reduced for CD45+ cellcontent in 11K2-treated versus MOPC21 (IgG1b control antisera)-treatedmice (50 cells/mm² versus about 120 cells/mm²).

ApoE −/− mice treated with 11K2 showed systemic abnormalities witheither early (5-17 weeks) or late treatment (17-29 weeks) with ananti-MCP antibody. On autopsy, mice treated with 11K2 antisera exhibitedno abnormalities relative to control animals in the following tissues:heart, liver, kidneys, lung, lymph nodes, brain, bone, skin, stomach,intestines, colon, salivary glands, gall bladder, prostate, testis,thymus, adrenals, pancreas, bladder, duodenum. No significantdifferences in CD3+, CD4+ and CD8+ levels were observed in blood, spleenand lymph node tissues of experimental apoE −/− mice treated with 11K2antisera relative to control-treated animals. Additionally, lipidprofiles of 11K2-treated mice were essentially identical tocontrol-treated mice when levels of total cholesterol, triglycerides,HDL cholesterol, and LDL cholesterol were examined.

Collagen and α-smooth muscle actin (ASMA) content were also assessed inplaques of 11K2-treated and control mice via immunolabelling. Plaquesfrom mice in both the early and late treatment groups that wereadministered control antisera MOPC21 exhibited collagen content of about3% for initial plaques, while advanced plaques of delayed treatmentgroup mice showed collagen contents of about 30%. In contrast, plaquesof early and late treatment mice administered 11K2 antisera showedsignificantly greater collagen content than control animals, as earlyand late treatment group initial plaques from 11K2-treated mice hadabout 6% collagen levels, and advanced plaques from delayed treatmentgroup mice treated with 11K2 exhibited about 40% collagen levels.Similar effects were observed for α-smooth muscle actin (ASMA). Plaquesfrom control-treated mice in both early and delayed treatment groupsexhibited about 0.5% ASMA levels for initial plaques, and advancedplaques from control-treated mice in the delayed treatment grouprevealed about 2% ASMA content. In contrast, plaques dissected from11K2-treated mice exhibited ASMA content of about 4% for early treatmentmice, and about 3% ASMA content for both initial and advanced plaquesdissected from delayed treatment mice.

Array-Based Detection of Differentially Expressed Genes inAtherosclerotic Mice

Gene array was used to examine the gene expression patterns ofatheroclerotic mice. Groups of C57BL/6 apoE −/− mice fed a diet ofnormal chow for 3, 4.5, or 6 months, or a western type diet for 3, 4.5,or 6 months, were sacrificed at the end of the experimental period,Following sacrifice, vascular tissue was dissected and subjected toarray-based expression profiling on mouse Unigene I arrays (IncyteGenomics, Inc.). Genes that were either upregulated or downregulated bygreater than two-fold in comparisons of apoE −/− mice on varying dietsto apoE −/− mice on normal chow diet for three months, were identifiedand examined. A preponderance of the differentially expressed genesidentified were involved in inflammation and fibrosis, including: thesmall inducible cytokines, such as MCP-1, MCP-2 and MIP; complementfactors; interleukins; cathepsins; MMP 2 and MMP 12; and TGF-β. A numberof small inducible cytokines were examined in more detail, includingFractalike (SIC D1), MIP 1 (SIC A3), MCP-1, MCP-2 (SIC A8), IL-8 like(SIC A6), MCP-3 (SIC A7), PDGF-inducible (SIC A2), and RANTES (SIC A5).When relative array-based expression data was examined for all of thesupra listed cytokines, all cytokines other than RANTES exhibitedincreases in relative expression levels as atherosclerosis progressed inboth normal chow diet and western type diet apoE −/− mice.

Analysis of MCP-1 RNA and protein levels revealed a marked increase inboth levels in ApoE −/− mice fed Wester chow. Elevated MCP-1 transcriptlevels during atherosclerosis progression produced correspondingincreases in levels of MCP-1 protein, as detected in aortic arch tissue.While MCP-1 protein was not detectable in serum for any mice examinedvia array-based expression profiling, MCP-1 levels of about 60 pg/mlwere observed for apoE −/− mice fed a western type diet for 4.5 months,and about 100 pg/ml levels of MCP-1 protein were observed for apoE −/−mice fed a western type diet for six months.

VI. Crystallization and Structural Determination of MCP-1-11K2-FabExample 30 Crystal Structure of 11K2

A three-dimensional structure of a complex of a Fab fragment of murine11K2 antibody with MCP-1 was determined by X-ray crystallography. TheFab fragment was produced by proteolytic cleavage. 1 mg of human MCP-1and 1 mg of murine 11K2 Fab was mixed and concentrated to 8 mg/ml. Equalvolumes of protein and well solution (10-15% PEG 4000, 100 mM HEPESpH=7.5, 30 mM glycl-glycl-glycine) were combined and placed at roomtemperature to equilibrate. Crystals appeared within 3 days and grew tofull size within 2 weeks. Crystals were flash-frozen for datacollection, using liquid nitrogen in a solution containing 100 mM HEPESpH=7.5, 15% PEG 4000, 30 mM glycl-glycl-glycine and 25% glycerol.Crystal transfer to the cryoprotection solution was necessary topreserve the crystal during cooling to −180° C. for data collection, andalso to allow compounds to bind under low salt conditions, which isimportant to increase the solubility of the compounds and to remove thesulfate ion bound in the active site (placed by crystallizationconditions, but removable upon soaking for several days).

Data collection was performed on a rotating anode X-ray'generator for aduration of typically about 6 hours, using 1 degree of oscillation and 5minute exposure times. An oscillation range of 60 degrees was requiredfor a complete data set. The space group of the crystals was determinedto be C222₁ with unit cell dimensions a=86.36 Å, b=89.10 Å, c=176.24 Å.In 22,774 unique reflections (542,403 total reflections), resolutionlimits were determined to be 50 to 2.5 Å (2.59-2.50 Å). Results from thedata collection are described below in Table 23.

TABLE 23 Data collection - phasing and model refinement Resolutionlimits: 50-2.5 Å (2.59-2.50 Å Number of total reflections: 542,403Number of unique reflections: 22,774 Redundancy: 6.2 I/o: 13.2 (3.3)R_(merge): 0.090 (0.436) Completeness: 99.8% (99.9%) R-factor/Rfree:0.219/0.277 # reflections for refinement: 23911Structure was solved by molecular replacement with Fab model. After aninitial round of refinement, MCP-1 was clearly visible in 2Fo-Fc maps.The final model contains residues 4-71 of MCP-1, 1-214 of the lightchain of 11K2, 1-217 of the heavy chain of 11K2, and 43 water molecules.Crystal structure results revealed that the residue contacts of MCP-1which the heavy chain of 11K2 binds include R30, T32, S34, K38, E39,V41, P55, K56, Q61, M64. Analysis of the crystal structure also revealedthat the light chain of 11K2 contacts residues D65, D68, K69 of MCP-1.Thus, 11K2 binds a discontinuous sequence of MCP-1.

Forming part of the present disclosure is the appended Sequence Listing,the contents of which are summarized in the table below:

TABLE 24 Summary of sequences SEQ ID Sequence NO: Description Type 1MCP-1 MRHAS motif amino acid 2 MCP-3 MRHAS motif amino acid 3 1A1 HeavyChain cDNA Primer nucleic acid 4 1A1 Light Chain cDNA Primer nucleicacid 5 1A1 Heavy Chain cDNA primer nucleic acid 6 1A1 Heavy Chain cDNAprimer nucleic acid 7 1A1 Light Chain cDNA primer nucleic acid 8 1A1Light Chain cDNA primer nucleic acid 9 1A1 Heavy chain variable regionnucleic acid 10 1A1 Light chain variable region nucleic acid 11 1A1Heavy chain variable region amino acid 12 1A1 Light chain variableregion amino acid 13 1A1 Heavy Chain Variable Region CDR1 amino acid 141A1 Heavy Chain Variable Region CDR2 amino acid 15 1A1 Heavy ChainVariable Region CDR3 amino acid 16 1A1 Light Chain Variable Region CDR1amino acid 17 1A1 Light Chain Variable Region CDR2 amino acid 18 1A1Light Chain Variable Region CDR3 amino acid 19 11K2 Heavy Chain cDNAPrimer nucleic acid 20 11K2 Light Chain cDNA Primer nucleic acid 21 11K2Heavy Chain cDNA Primer nucleic acid 22 11K2 Heavy Chain cDNA Primernucleic acid 23 11K2 Light Chain cDNA Primer nucleic acid 24 11K2 LightChain cDNA Primer nucleic acid 25 11K2 Heavy Chain Variable Regionnucleic acid 26 11K2 Light Chain Variable Region nucleic acid 27 11K2heavy chain variable region amino acid 28 11K2 light chain variableregion amino acid 29 11K2 Heavy Chain Variable Region CDR1 amino acid 3011K2 Heavy Chain Variable Region CDR2 amino acid 31 11K2 Heavy ChainVariable Region CDR3 amino acid 32 11K2 Light Chain Variable Region CDR1amino acid 33 11K2 Light Chain Variable Region CDR2 amino acid 34 11K2Light Chain Variable Region CDR3 amino acid 35 11K2 heavy chain chimeranucleic acid 36 11K2 light chain chimera nucleic acid 37 11K2 heavychain chimera amino acid 38 11K2 light chain chimera amino acid 39 11K2humanized heavy chain, version 1 nucleic acid (includes constant region)40 11K2 humanized heavy chain, version 1 amino acid (includes constantregion) 41 11K2 humanized heavy chain, version 2 nucleic acid (includesconstant region) 42 11K2 humanized heavy chain, version 2 amino acid(includes constant region) 43 11K2 humanized light chain, version 1nucleic acid (includes constant region) 44 11K2 humanized light chain,version amino acid 1(includes constant region) 45 11K2 humanized lightchain, version 2 nucleic acid (includes constant region) 46 11K2humanized light chain, version 2 amino acid (includes constant region)47 Humanized 11K2 heavy chain, variable, amino acid version 1 48Humanized 11K2 heavy chain, variable, v2 amino acid 49 Humanized 11K2light chain, variable, v1 amino acid 50 Humanized 11K2 light chain,variable, v2 amino acid 51 Chimera, variable heavy chain 1A1 amino acid52 Chimera, variable light chain 1A1 amino acid 53 Humanized 1A1 heavychain, variable v1 amino acid 54 Humanized 1A1 heavy chain, variable v2amino acid 55 Humanized 1A1 light chain, variable v1 amino acid 56Humanized 1A1 light chain, variable v2 amino acid 57 v1 light chainprimer nucleic acid 58 v1 light chain primer nucleic acid 59 v1 lightchain primer nucleic acid

1-50. (canceled)
 51. An isolated nucleic acid molecule encoding a heavychain comprising the complementarity determining regions as set forth inSEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31, wherein the remainder ofthe heavy chain is from a human immunoglobulin.
 52. An isolated nucleicacid molecule encoding a light chain comprising the complementaritydetermining regions as set forth in SEQ ID NO:32, SEQ ID NO:33, and SEQID NO:34, wherein the remainder of the light chain is from a humanimmunoglobulin.
 53. An isolated nucleic acid molecule encoding ahumanized immunoglobulin or antigen-binding fragment thereof comprisinga) heavy chain complementarity determining regions as set forth in SEQID NO:29, SEQ ID NO:30, and SEQ ID NO:31, and b) light chaincomplementarity determining regions as set forth in SEQ ID NO:32, SEQ IDNO:33, and SEQ ID NO:34 wherein the remainder of the immunoglobulin orantigen-binding fragment is from a human immunoglobulin.
 54. An isolatednucleic acid molecule comprising a nucleotide sequence corresponding tothe amino acid sequence selected from the group consisting of SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, and SEQ ID NO:50. 55-56. (canceled) 57.A vector comprising the nucleic acid molecule of claim
 53. 58.(canceled)
 59. A host cell comprising the nucleic acid molecule of claim53.
 60. The host cell of claim 59, wherein the cell is mammalian. 61.The host cell of claim 59, wherein the cell is bacterial. 62-69.(canceled)
 70. The nucleic acid of claim 51, wherein the heavy chainfurther comprises variable region framework residues L27, I29, and T73(Kabat numbering convention) from the monoclonal antibody 11K2 heavychain as set forth in SEQ ID NO:27.
 71. The nucleic acid of claim 51,wherein the heavy chain further comprises at least one variable regionframework residue selected from the group consisting of N28, K30, I48,and A67 (Kabat numbering convention) from the monoclonal antibody 11K2heavy chain as set forth in SEQ ID NO:27.
 72. The nucleic acid of claim71, wherein the heavy chain further comprises variable region frameworkresidues N28, K30, I48, and A67 (Kabat numbering convention) from themonoclonal antibody 11K2 heavy chain as set forth in SEQ ID NO:27. 73.The nucleic acid of claim 52, wherein the light chain further comprisesat least one variable region framework residue from the monoclonalantibody 11K2 light chain set forth as SEQ ID NO: 28, wherein theresidue is selected from the group consisting of S49 and Y71 (Kabatnumbering convention).
 74. The nucleic acid of claim 52, wherein thelight chain further comprises variable region framework residues S49 andY71 (Kabat numbering convention) from the monoclonal antibody 11K2 lightchain as set forth in SEQ ID NO:28.
 75. The nucleic acid of claim 52,wherein the light chain further comprises variable region frameworkresidue K69 (Kabat numbering convention) from the monoclonal antibody11K2 light chain as set forth in SEQ ID NO:28.
 76. The nucleic acid ofclaim 51, wherein the heavy chain further comprises at least onevariable region framework residue from the monoclonal antibody 11K2heavy chain set forth as SEQ ID NO: 27, wherein the residue is selectedfrom the group consisting of L27, N28, I29, K30, I48, A67, and T73(Kabat numbering convention).
 77. The nucleic acid of claim 76, whereinthe heavy chain comprises variable region framework residues L27, N28,I29, K30, I48, A67, and T73 (Kabat numbering convention) from themonoclonal antibody 11K2 heavy chain as set forth in SEQ ID NO:27. 78.The nucleic acid of claim 76, wherein the heavy chain comprises at leastone variable region framework residues L27, I29, and T73 (Kabatnumbering convention) from the monoclonal antibody 11K2 heavy chain asset forth in SEQ ID NO:27.
 79. The nucleic acid of claim 52, wherein thelight chain further comprises at least one variable region frameworkresidue from the monoclonal antibody 11K2 light chain set forth as SEQID NO: 28, wherein the residue is selected from the group consisting ofS49, K69, and Y71 (Kabat numbering convention).
 80. The nucleic acid ofclaim 53, wherein the heavy chain further comprises at least onevariable region framework residue from the monoclonal antibody 11K2heavy chain set forth as SEQ ID NO: 27, wherein the residue is selectedfrom the group consisting of L27, N28, I29, K30, I48, A67 and T73 (Kabatnumbering convention), and the light chain comprises at least onevariable region framework residue from the monoclonal antibody 11K2light chain set forth as SEQ ID NO: 28, wherein the residue is selectedfrom the group consisting of S49, K69, and Y71 (Kabat numberingconvention).
 81. The nucleic acid of claim 80, wherein the heavy chaincomprises variable region framework residues L27, I29, and 173 (Kabatnumbering convention), and the light chain comprises variable regionframework residues S49 and Y71 (Kabat numbering convention).
 82. Thenucleic acid of claim 80, wherein the heavy chain comprises variableregion framework residues L27, I29, and T73 (Kabat numberingconvention), and the light chain comprises variable region frameworkresidues S49, K69, and Y71 (Kabat numbering convention).
 83. The nucleicacid of claim 80, wherein the heavy chain comprises variable regionframework residues L27, N28, I29, K30, I48, A67 and T73 (Kabat numberingconvention), and the light chain comprises variable region frameworkresidues S49 and Y71 (Kabat numbering convention).
 84. The isolatednucleic acid molecule encoding a humanized immunoglobulin orantigen-binding fragment of claim 53 wherein the heavy chain furthercomprises variable region framework residues L27, N28, I29, K30, I48,A67, and T73 (Kabat numbering convention) from the monoclonal antibody11K2 heavy chain set forth as SEQ ID NO: 27, and the light chain furthercomprises variable region framework residues S49, K69, and Y71 (Kabatnumbering convention) from the monoclonal antibody 11K2 light chain setforth as SEQ ID NO: 28, wherein the remainder of the heavy and lightchains are from a human immunoglobulin.